Correspondence: Chu-Long Zhang, State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China. Tel.: +86 571 8697 1185; fax: +86 571 8697 1516; e-mail: email@example.com
A survey of the endophytic fungal community of wild rice (Oryza granulata) in China was conducted. Two isolates recovered from healthy roots are assumed to be dark septate endophytes (DSEs). They are morphologically similar to species from the genus Harpophora and are identified as a new species, Harpophora oryzae, based on the molecular phylogeny and morphological characteristics. A neighbor-joining tree constructed from ITS–5.8S rRNA gene regions reveals that H. oryzae forms a distinctive subclade within the genus Harpophora, and is not genetically close to other species of Harpophora. Harpophora oryzae exhibits a moderate growth rate, with a frequent production of rope-like strands. It sporulates readily on artificial medium. Phialides are usually flask or bottle shaped and occur singly along hyphae or laterally and terminally on branched, hyaline to brown conidiophores, and also form whorls on metulae. Conidiophores are mostly branched with a slightly thickened wall, varying in dimensions. Conidia are one-celled and hyaline, most of them being falcate and strongly curved. The morphological differences between Harpophora spp. and Harpophora-like anamorphs representing different orders are also discussed. An in vitro inoculation test showed that H. oryzae may contribute towards improving rice (Oryza sativa L.) growth. Microscopic inspection of roots and phylogenetic placement of isolates further confirmed that H. oryzae represents a novel member of DSEs.
Mycelium radicis atrovirens or dark septate endophytes (DSE) are a phylogenetically diverse group among root fungal endophytes (Sieber, 2002; Grünig et al., 2008). These fungi are generally characterized by melanized, septate hyphae and do not readily sporulate in artificial media. The Phialophora–Gaeumannomyces complex and Phialocephala fortinii constitute two major subgroups of DSEs (Sieber, 2002). Certain members of the genera Phialophora, now Harpophora spp., usually live in herbaceous plant roots as hosts, especially in Gramineae (Sieber, 2002). The genus Phialophora s.l. is also traditionally considered to be highly polymorphic (Jumpponen & Trappe, 1998; Gams, 2000). Likewise, there are still some disagreements between the morphological and the molecular identification of Phialophora spp. (Yan et al., 1995; de Hoog et al., 1999; Ulrich et al., 2000; Sieber, 2002). Species formerly classified in the genus are now known to belong to different orders of Ascomycetes.
Gams (2000) began to sort out the taxonomy of Phialophora spp. and erected Harpophora for anamorphs of Gaeumannomyces and Magnaporthe within the Magnaporthaceae. Its morphological characteristics include fast-growing, thin colonies with ‘runner hyphae’ and more or less pigmented phialides coupled with cylindrical, hyaline and strongly curved conidia. Up to now, four species combinations have been described within Harpophora, i.e. Harpophora radicicola (type species, previously Phialophora radicicola) (McKeen, 1952; Walker, 1980), Harpophora maydis (Cephalosporium maydis) (Samra et al., 1963), Harpophora graminicola (Phialophora graminicola) (Hornby et al., 1977; Walker, 1980) and Harpophora zeicola (Phialophora zeicola) (Deacon & Scott, 1983). In addition, the anamorphs of Gaeumannomyces spp. belong here without being separately named as anamorph species.
We have recently started an examination of the endophytic fungal community in wild rice (Oryza granulata) roots in China, during which we found a new species, which is described here as Harpophora oryzae.
Materials and methods
Sites of study, sampling, fungi isolation, purification and storage of endophytic cultures
The site of study is located in Xishuangbanna, Yunnan province, southwest of China (22°04′–22°17′N; 100°32′–100°44′E). In September of 2007 and 2008, we collected samples from two sites in Xishuangbanna. Healthy and intact wild rice plants with bulk soil were packed in a box and carefully transported to the laboratory within 48 h.
For isolation of endophytic fungi, healthy roots (free of detectable lesions) of the sample rice plants were gently rinsed with tap water, immersed in ethanol (75% v/v) for 30 s, then in sodium hypochlorite (1% w/v) for 10 min and finally rinsed three times in sterile-distilled water. Roots were cut into segments of 0.6 cm length and transferred to a malt extract agar (MEA) plate containing 2% malt extract and 2% agar (w/v) supplemented with chloramphenicol (50 mg L−1) to prevent bacterial growth. Six root fragments were placed on one plate and incubated at 25 °C in permanent darkness. After the emergence of fungal hyphae, these were cut off and subcultured. Isolates were stored by covering a culture on potato dextrose agar (PDA) slants with sterile liquid paraffin at 25 °C and by preservation in aqueous 15% v/v glycerol additionally containing glucose (10 g L−1), yeast extract (1 g L−1) and casein hydrolysate (1 g L−1) at −70 °C.
Light-microscopic analysis was performed using an Olympus BX51 microscope. Images were acquired using axiovision 3.1. For the determination of spore characteristics, specimens were mounted in water. The colony appearance and growth rates were determined on PDA and MEA in the dark at 25 °C for a period of 7 days.
Scanning electronic microscopy was conducted using cryo-SEM (Hitachi S-3000N microscope, Japan), operating between 10 and 15 kV on samples containing a thin layer of gold sputter coating. Strain R5-6-1 was cultivated on PDA medium for 5 days in the dark at 25 °C, during which time conidiophore and conidia formation started. The margin of the culture was then sliced out. The operation was carried out carefully not to deform the surface features of the culture.
Fungal DNA extraction, PCR, sequencing and phylogenetic analysis
The fungal strain was cultured in PD broth for 4 days at 180 r.p.m. min−1 in an orbital shaker at 25 °C. Fungal DNA was extracted using the Multisource Genomic DNA Miniprep Kit (Axygen Bioscience, Inc.) following the manufacturer's instructions. Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990) were used for amplification of the fungal rDNA internal transcribed spacer (ITS) regions 1 and 2. The PCR reaction (50 μL total volume) contained 5 μL 10 × PCR buffer, 7 μL 25 mM Mg2+, 2 μL 2.5 mM dNTP, 2 μL of each primer (10 μM), 4 μL (0.5–10.0 ng) of total DNA, 1 μL Taq polymerase and 27 μL ddH2O. Thirty-five cycles were run, each consisting of a denaturation step at 94 °C (40 s), an annealing step at 54 °C (50 s) and an extension step at 72 °C (60 s). After the 35th cycle, a final 10-min extension step at 72 °C was performed. The reaction products were separated in a 1.0% w/v agarose gel and bands were stained with ethidium bromide. The PCR products were then purified using the DNA Gel Extraction Kit (Axygen Bioscience, Inc.) and sequenced in an ABI 3730 sequencer (Applied Biosystems) using the ITS1 and ITS4 primers. The sequences were subjected to a blast search and were aligned using clustal x together with the next neighbors (i.e. sequences that had a negative probability e-value of 0.0 in a blast search against the GenBank database); the alignment was manually corrected in genedoc. The evolutionary distance was determined using the Jukes–Cantor model to construct a phylogenetic tree by the neighbor-joining method using phylogeny inference package (phylip, v 3.68). The resultant trees were analyzed using the program consense to calculate a majority rule consensus tree. The tree file was then displayed by treeview. Bootstrap (1000 replicates) analysis used SEOBOOT, DNADIST, NEIGHBOR and CONSENSE in phylip.
Molecular phylogenetic analysis of Harpophora and other related genera in Magnaporthaceae
Sequence inspection of the ITS1, 5.8S rRNA gene and ITS2 regions showed 100% identity of H. oryzae isolates R5-6-1 and RC-3-1. The blast similarity search revealed that H. oryzae shared 96%, 95% and 95% identity with ITS 1 and 2 sequences of unidentified Harpophora spp. (AJ132541), Harpophora spp. (AJ132542) and Harpophora spp. (AJ010039), respectively. In order to relate H. oryzae to already known Harpophora sequences and species and other related genera in Magnaporthaceae, a phylogenetic analysis was performed. As shown in Fig. 1, the NJ tree grouped Harpophora spp. into four clades (A, B, C and D), which were strongly supported with high bootstrap values. Among these, H. oryzae forms a well-supported distinct sister group in clade B, which also contained three other so far unnamed Harpophora spp. (anamorphs of Gaeumannomyces) and two isolates of Buergenerula spartinae. Harpophora zeicola, H. radicicola and Gaeumannomyces graminis and its anamorph are clustered in clade A; species of Gaeumannomyces amomi and Pyricularia zingiberis were also clustered into this clade. Gaeumannomyces cylindrosporus and its assumed anamorph H. graminicola formed clade C; and H. maydis constituted clade D.
Fungus endophyticus in radicibus Oryzae granulata. Coloniae in agaro PDA olivaceo-brunneae, velutinae. Hyphae aeriae 2.0–3.5 μm latae, hyalinae vel brunneae. Conidiophora solitaria, interdum pauca fasciculata, simplicia, laxe ramosa, brunnea. Phialides solitares in hyphis et saepe terminales in conidiophoris, 2–4 fasciculatae, lageniformes, brunneae, 5.5–14 × 2.5–3 μm. Conidia in capitulis mucosis aggregata, hyalina, continua, falcata, conspicue curvata, laeves, 7.5–9 × 0.8–1.2 μm.
Colony diameter approximately 4.5 cm on MEA or PDA in the dark after 7 days at 25 °C. Aerial mycelium denser on MEA than on PDA. Rope-like strands formed by wavy hyphae. Colony color gray-olivaceous first, then becoming fuscous in old cultures and forming dense and gray felt of aerial mycelium on PDA, conidia produced abundantly (Fig. 2a–c). Colony reverses, turning gray-olivaceous. Aerial hyphae septate, 2.0–3.5 μm wide, hyaline to brown. Conidiophores unbranched or branched 1–2 times with a slightly thickened wall, mostly arising singly, sometimes fasciculate, bi- to terverticillate, varying in dimensions, with a range of 15–110 × 2.8–5 μm. Metulae one to three per branch, two to four phialides per metula. Phialides occurring singly along hyphae or laterally and terminally on branched, hyaline to brown conidiophores, usually forming whorls on the metulae, flask or bottle shaped, 5.5–14 μm long (n=15), 2.5–3 μm wide at the widest point, 1.5–2.0 μm wide at the base, collarette 0.5–1.2 μm wide (n=10), pale brown to brown. Conidia accumulated in slimy heads on the tips of phialides, hyaline, unicellular, falcate, strongly curved, 7.5–9 μm long (along the curvature of the conidia), 0.8–1.2 μm wide at the widest point (n=20) (Figs 3a, b, 4 and 5). Intercalary chlamydospores, obovoid to ellipsoid, occasionally in chains.
Habitat and distribution: Endophytic in healthy roots of O. granulata. Known from South-West China.
Holotype: China, Xishuangbanna, National Nabanhe river reserve, isolated from root tissues of wild rice seedlings, 27/09/2007, Z.L. Yuan; lyophilized culture no. R5-6-1 was deposited at Centraalbureau voor Schimmelcultures (CBS 125863) and China General Microbiological Culture Collection Center (CGMCC 2737). Additional cultures were deposited at the State Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, China. The ITS sequences of two isolates of H. oryzae have been submitted to the GenBank database with the accession numbers EU636699 (R5-6-1) and FJ752606 (RC-3-1).
Dematiaceous septate fungi are well known as important components of the fungal consortium that colonizes plant roots. Among them, Phialocephala spp. and Phialophora spp. are well-recognized members. In particular, Phialophora spp. preferentially reside in grass roots systems, and display pathogenic or mutualistic relationships with their hosts (Newsham, 1999; Sieber, 2002; Mandayam & Jumpponen, 2005; Sieber & Grünig, 2006), while Phialophora finlandica (now called Cadophora finlandica) has been shown to form ectendomycorrhizae with a variety of woody plants (Wang & Wilcox, 1985).
Phialophora was first introduced by Medlar (1915) with Phialophora verrucosa as a type (de Hoog et al., 1999), which belongs to the Herpotrichiellaceae in the Chaetothyriales. As documented above, the Phialophora genus has been poorly defined with vaguely morphological descriptions. Therefore, a subdivision of Phialophora-like divergent anamorph groups would be necessary. Considerable efforts have been made to clarify the taxonomy of little differentiated Phialophora-like fungi. For example, P. finlandica, Phialophora gregata and Phialophora malorum are now placed into the Cadophora genus (Harrington & McNew, 2003); a new anamorph genus, Pleurostomophora, is now proposed to accommodate two species of Phialophora (Phialophora repens and Phialophora richardsiae) (Vijaykrishna et al., 2004); the Phaeoacremonium genus was also erected to accommodate formerly described Phialophora parasitica (Crous et al., 1996); and the Lecythophora genus was reintroduced to accommodate P. hoffmannii (Gams & McGinnis, 1983). The Harpophora genus is also thus introduced to classify the Phialophora anamorph of Gaeumannomyces and Magnaporthe, which is recognized as a monophyletic group (Gams, 2000). All the above rearrangements of Phialophora-like fungi were based on morphological examinations and the molecular phylogeny of nuclear rDNA regions (LSU and/or ITS). Saleh & Leslie (2004) confirmed that C. maydis fell within the Gaeumannomyces–Harpophora spp. complex and supported its classification as H. maydis with an integrated analysis of ITS, β-tubulin and histone H3 sequences. Our molecular data also support that all identified Harpophora spp. are clustered in the Gaeumannomyces group and H. oryzae forms a distinct clade, which is clearly separated from other Harpophora spp. In addition, it is clearly demonstrated that P. zingiberis, G. amomi and B. spartinae also appear to be related closely to the Gaeumannomyces–Harpophora complex, while Pyricularia longispora and Magnaporthe salvinii occur separately (Fig. 1), which is in accordance with other previous studies on the molecular phylogeny in Magnaporthaceae (Bryan et al., 1995; Bussaban et al., 2005; Huhndorf et al., 2008; Thongkantha et al., 2009). However, B. spartinae and three Harpophora isolates and two isolates of H. oryzae are clustered with very low bootstrap value support (<50%) (Fig. 1).
Harpophora spp. with Gaeumannomyces teleomorphs are well known as causes of take-all diseases of wheat and grasses (Freeman & Ward, 2004). Although H. oryzae is a close relative of Gaeumannomyces, an in vitro pathogenicity test shows that H. oryzae acts as a nonpathogenic endophyte colonizing cultivated rice (Oryza sativa L.) roots. Intracellular hyphae are found in the root cortex. After 30 days of coculture in half-strength Murashige and Skoog (1/2 MS) medium under aseptic conditions (25 °C, 18 h light/6 h darkness), H. oryzae strongly promotes growth and biomass formation of rice plants (see Supporting Information, Figs S1 and S2), similar to H. graminicola, a beneficial DSE of grasses (Kirk & Deacon, 1987; Newsham, 1999). In previous reports, isolates of the naturally occurring nonpathogenic G. cylindrosporus were effective in controlling talk-all when introduced into wheat crops (Gutteridge et al., 2007).
Fungi living as endophytes in wild rice have not yet been reported. During our search in 2007 and 2008, we recovered two Phialophora-like fungal isolates from 354 samples in healthy roots, indicating a very low isolation rate. The present paper introduces H. oryzae as one of probably many other endophytes in this important crop plant. Based on the morphological characteristics, we place our novel isolates in Harpophora. We were unable to observe a teleomorph of these two isolates; also, keeping the two cultures for 3 weeks on oatmeal agar under light did not lead to fruiting body formation. To our knowledge, no Harpophora spp. has so far been found to be associated with cultivated rice plants (Fisher & Petrini, 1992; Tian et al., 2004; Naik et al., 2009; Vallino et al., 2009), but one recovered isolate was identified as P. verrucosa (Naik et al., 2009).
Three Harpophora isolates recovered from wheat and barley in Germany and the United Kingdom (Ward & Bateman, 1999; Ulrich et al., 2000) (accession numbers: AJ132541, AJ132542 and AJ010039) formed a sister subclade to H. oryzae. It is possible that these are also H. oryzae or an allopatric species to it. Unfortunately, the three strains were not available for this study, and thus this question could not be answered. Hence, we have examined only the morphological description of the currently identified Harpophora spp. Harpophora oryzae is shown to be morphologically similar to H. zeicola, a maize root parasite (Deacon & Scott, 1983), and H. graminicola. It differed from H. zeicola in having massive aggregations of falcate conidia and densely branched conidiophores. Harpophora zeicola produced two types of conidia, one of which resembled those of H. oryzae; in H. oryzae, phialides are almost straight, while they are often curved in H. zeicola. The major differentiation from H. graminicola is in the conidial morphology. A comprehensive comparison of phenotypic characteristics of H. oryzae with four close relatives is presented in Table 1.
Table 1. Comparison of the morphological characteristics of Harpophora oryzae with other close relatives
Conidia morphology and dimension
Colony characteristic and growth rate
H. oryzae sp. nov.
Little variable, almost strongly curved and sickle-shaped, single-celled; 7.5–9.0 × 0.8–1.2 μm
Mostly in large clusters and densely branched; straight, sometimes curved; more or less pigmented; funnel-shaped collarets, often incurved at the open end
Gray-olivaceous first, then becoming fuscous in old culture; hyphal ropes in the margin of the colony, moderate growth
Wild rice roots
Cylindrical, strongly curved and hyaline, single-celled; 4.1–9.6 × 0.7–1.5 μm.
More or less pigmented, often curved with flared collarettes
More or less olivaceous-brown, broader radiating ‘runner hyphae’, growing rapidly
Very variable, almost straight, sometimes curved or even sigmoid, single-celled; 2.9–6.3 × 0.7–2.0 μm.
Very variable, flask-shaped, straight or curved, sometimes with flared collarettes
Olivaceous, moderate growth
Vulpia ciliata and wheat roots
Straight and oblong, single-celled; 3.6–14 × 3–3.6 μm.
Hyaline, mostly branched and straight, inconspicuous collarettes
White to pale gray or becoming slate gray with age, slow growing
Maize roots and stems
Two types of conidia: one type is narrow shaped and strongly curved, the other type is larger and rounded, single-celled; 6–20 × 1.5–6 μm and 5–9 × 1–1.5 μm.
Mostly in large clusters; irregular, often curved, sometimes sigmoid; inconspicuous to funnel-shaped collarettes
White first, then becoming grayish brown, growing rapidly
Morphologically, the new erected genera for accommodating some previously described Phialophora-like ascomycetes including Phaeoacremonium (Magnaporthaceae), Pleurostoma (Calosphaeriales) and true Phialophora (Chaetothyriales) are also shown to be different from Harpophora when compared with their morphology of phialides and conidia, and the pigmentation of the mycelium (Gams, 2000; Vijaykrishna et al., 2004; Mostert et al., 2006). Gams (2000) therefore listed a series of important criteria for the subdivision of phialidic hyphomycetous species with more or less pigmented mycelium.
Collectively, based on ITS sequence-based phylogeny and comparison of the morphological characteristics, we consider it safe to introduce H. oryzae as a new species of Harpophora. The molecular and physiological interactive mechanisms with respect to H. oryzae–rice association are being studied.
This work was supported by the National Natural Science Foundation of China (Grant No. 30600002 and 30970097) to C.-L.Z. We would like to thank Walter M. Jaklitsch (Vienna University of Technology, Austria) for improving the Latin species description.