This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, Y.-C. Articles by Cheng, T.-Y. Search for Related Content PubMed Articles by Chen, Y.-C. Articles by Cheng, T.-Y. Agricola Articles by Chen, Y.-C. Articles by Cheng, T.-Y.

Caecomyces sympodialis sp. nov., a new rumen fungus isolated from Bos indicus

     Institute of Biotechnology, National Pingtung University of Science and Technology, Neipu Hsiang, Pingtung, 91201 Taiwan, R.O.C.

Chiu-Yuan Chien

     Department of Life Sciences, National Taiwan Normal University, Taipei, 10677 Taiwan, R.O.C.

Chun-Yi Hu

     Department of Nutrition and Health Science, Fooyin University, Taliao Hsiang, Kaohsiung, 83161 Taiwan, R.O.C.

Tai-Yi Cheng

     Yung-Ta Institute of Technology and Commerce, Pingtung, 90901 Taiwan, R.O.C.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY AND DISCUSSION LITERATURE CITED

 

A new anaerobic rumen fungus was isolated from the rumen fluid of a yellow cow (Bos indicus). This fungus appears to be a previously undescribed species of the genus Caecomyces, it possessing uniflagellate zoospores, a spherical holdfast, tubular sporangiophores and bulbous rhizoids. This new fungus also features distinctive multisporangiate thallus sympodially distributed on sporangiophores. The fungus resembles Caecomyces communis and C. equi in that it characterizes bulbous rhizoids and uniflagellate zoospores but differs from C. communis and C. equi in that it possesses multisporangiate and sympodial sporangia. This new fungus and Cyllamyces aberensis both reveal similar morphology during early thallus development in having a spherical holdfast, but they vary from unbranched sporangiophores and additional bulbous rhizoids. In addition, the molecular phylogenetic analyses ITS1 (internal transcribed spacer 1) also conform to the results of the morphological examinations of Caecomyces. For the mentioned reasons, this new species of fungus is described as Caecomyces sympodialis sp. nov. The genera of Neocallimasticaceae and species of Caecomyces are also keyed out.

Key words: anaerobic fungus, Bos indicus, ITS1 sequence analysis, Neocallimasticaceae

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY AND DISCUSSION LITERATURE CITED

 
Anaerobic rumen fungi commonly survive in the gastrointestinal tract of herbivores. They are indispensable for the digestion of ruminants and play a major role in plant cell-wall decomposition (Fonty et al 1990, Trinci et al 1994). The first known anaerobic rumen fungus, Neocallimastix frontalis

Orpin, was confirmed and proposed by Orpin (1975). These rumen fungi were assigned to the order Neocallimasticales of Chytridiomycota based on its morphological characteristics and ribosomal DNA analysis (Li et al 1992, 1993). The family Neocallimasticaceae comprises six genera including Neocallimastix, Piromyces, Orpinomyces, Anaeromyces, Caecomyces and Cyllamyces (Ho and Barr 1995, Ozkose et al 2001). Among these, members of Caecomyces can be observed to be unique morphologically, distinguished from other genera of rumen fungi by the presence of bulbous rhizoids (Gold et al 1988). A rumen fungus featuring bulbous rhizoids first was reported by Orpin (1975), and the isolate was nominated as Sphaeromonas communis sensu Orpin (Orpin 1976); however, it is not a validly published name and was excluded specifically. The description of S. communis subsequently was amended, supplied a Latin diagnosis and cited as a Caecomyces communis comb. nov. by Gold et al (1988) (Ho and Barr 1995, Wubah and Fuller 1991). Thereat, C. equi and C. communis were found and classified in the genus Caecomyces. These two species mainly can be distinguished from each other by the number of bulbous rhizoids. One of these two species, C. equi, develops a single bulbous rhizoid on each thallus. The other species in the genus Caecomyces, C. communis, features a thallus that typically develops two or more bulbous rhizoids when mature. In addition the monocentric thallus of C. equi develops one sporangium, whereas the thallus of C. communis occasionally possesses 2–4 sporangia (Gold et al 1988, Ho and Barr 1995).

In this study, we discuss the features of a new species, Caecomyces sympodialis, that is unique among the genus Caecomyces in producing sympodial multisporangiate sporangia and unbranched tubular sporangiophores. These morphological features distinguish it from previously known species of Caecomyces.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY AND DISCUSSION LITERATURE CITED

 
Isolation and cultivation.— – Rumen fluid was collected from a yellow cow (Bos indicus). The fluid was sampled through a cannula and syringed into a Hungate-type anaerobic culture tube (125 x 16 mm, Bellco Glass Inc., USA) containing 5 mL isolation agar medium (Chen et al 2002). The protocols for rumen fungus isolation were as those described by Chen et al (2002); the retained isolate was designated W101. Cultures were maintained by subculturing every 3 d using isolation broth with a filter paper strip (60 x 10 mm, Whatman No. 1) as the sole carbon source. To suppress bacterial growth, 1.2% (w/v) penicillinG, 0.265% (w/v) streptomycin and 0.06% (w/v) chloramphenicol were used as antibiotics (Chen et al 2002). For reference purposes, Caecomyces communis CY50 also was obtained from the same animal by the same protocols described above.

Mycological observations and carbon-source utilization.— – W101 isolate cultured on isolation broth with carbon sources, such as 0.5% (w/v) glucose (16325, Riedel-de Haën, Germany), 0.5% (w/v) cellulobiose (C-7252, Sigma Chemical Co., USA ), filter paper (10 x 60 mm, Whatman No. 1), 0.2% (w/v) carboxylmethyl cellulose (C-5678, Sigma Chemical Co. USA), 0.5% (w/v) xylan (X-0627, Sigma Chemical Co., USA) and rice straw, were examined morphologically under a light microscope (CX21, Olympus Imaging Corp., Japan) and photographed by an attached digital camera (C-5050, Olympus Inc. Japan). Microscopic slides were prepared at various growth stages of isolate W101, stained with phloxin red (P-2759, Sigma Chemical Co., USA) and mounted in lactophenol for subsequent light microscope observation. For the observation of nuclei, a drop of cell suspension was mixed with 2% (w/v) glutaraldehyde (G-6257, Sigma Chemical Co., USA) in PBS (phosphate buffered saline) with 0.1% (w/v) DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Wako Chemical Co., Japan). A fluorescence microscope (BX40, Olympus Imaging Corp., Japan) with DAPI exciter filter was used for nucleus observation (Chen et al 2002). An isolate of Caecomyces communis CY50 was examined for morphology of zoospores and vegetative thallus including bulbous rhizoids, sporangiophores and sporangia according to the procedure of Wubah and Fuller (1991).

Phylogenetic analyses.— – Genomic DNA extraction, PCR amplification and sequencing were performed as outlined by Chen et al (2003). Sequences of ITS region 1 for isolates W101 and CY50 and the reference sequences (TABLE I), which were accessed from GenBank (National Center for Biotechnology Information, NIH, Bethesda, Maryland), were aligned with BioEdit software (Hall 1999). To minimize the number of gap, the alignment was checked by eye and corrected manually. Maximum parsimony and minimum evolution analyses were performed by MEGA software (Kumar et al 2004) compiled for the Microsoft Windows XP-compatible PC platform. In maximum parsimony analysis, the option "close-neighbor intercharge (CNI) with search level 1." was in effect. The consensus tree was generated by 1000 bootstraps replicated with two random addition trees for each replicate. The minimum evolution result was obtained by the neighbor joining method featuring 1000 bootstrap replication (Brookman et al 2000, Retief 2000).

	TAXONOMY AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY AND DISCUSSION LITERATURE CITED

View larger version (124K): [in this window] [in a new window]   FIGS. 1–9. Development of Caecomyces sympodialis isolate W101. 1. A uniflagellate zoospore, bar = 10 µm. 2. A sporangiophore arising from the holdfast, bar = 10 µm. 3. An incipient vegetative thallus and a bulbous rhizoid arising from the holdfast, bar = 10 µm. 4–6. The growth and development of a vegetative thallus, more than one sporangia can be seen arising from a sporangiophore. bar = 10 µm. 7. A mature thallus and sporangia arising from sporangiophores extending from a holdfast. h: holdfast, f: sporangiophore, r: rhizoid, sp: sporangium, bar = 10 µm. 8. Mature thalli with bulbous rhizoids and sporangia arising from three distinct unbranched sporangiophores. A sporangium with a septum formed at its base can be seen, bar = 10 µm. 9. Multisporangiate thalli attached onto the cellulose fragments.

Rhizoids bulbous. Sporangiophores tubular or unbranched. Multisporangiate sporangia frequently spherical. Zoospores uniflagellate or rare quadriflagellatae. Obligate anaerobe.

TYPE SPECIES: Dried specimen has been deposited in the herbarium of TNM (TNM F19502).

Etymology. – Referring the sporangia sympodially developed on the sporangiophores.

Habitat. – The rumen of Bos indicus.

Morphological observation and life cycle.— – The zoospores of Caecomyces sympodialis W101 were spherical to ovoid (FIG. 1), 5.0 ± 1.1 µm (n = 84) diam and possessed a single posteriorly flagellum 24.3 ± 4.7 µm (n = 84) long (FIG. 1), although zoospores occasionally were observed to be biflagellate (n = 2) and even quadriflagellate (n = 1). Such variation also has been described as being present in other uniflagellate rumen fungi (Ho and Barr 1995). After a period of time, zoospores became inactive and the flagellum became detached and scattered before encystment. The vegetative thallus of isolate W101 germinated endogenously from a cyst and subsequently formed a spherical holdfast (Ozkose et al 2001, Wubah and Fuller 1991). Afterward, one or two bulbous rhizoids arose, several sporangiophores emerged and extended from a holdfast and developed an incipient vegetative thallus (FIGS. 2–3). These sporangiophores usually were unbranched and tubular, with the young spherical sporangia growing sympodially on the terminal or middle segment of the sporangiophores (FIGS. 4–6). The average diameter of young sporangia, rhizoids and holdfasts was 6.8 ± 1.3 µm (n = 62), 7.7 ± 1.4 µm (n = 34) and 14.1 ± 3.7 µm (n = 22), respectively. The holdfast and tubular sporangiophores continuously grew and extended, and the incipient sporangia also developed and expanded progressively. A mature multisporangiate thallus formed at 48–72 h (FIGS. 7–8). Multisporangiate thalli often were noted to clump together or to attach onto the debris of the filter paper (FIG. 9) and the inner surface of the Hungate tube.

The bulbous rhizoids and holdfasts of mature multisporangiate thalli were spherical to ovoid, 8.6 ± 3.2 µm (n = 28) and 18.3 ± 3.7 µm (n = 20) diam, respectively. Multisporangiate sporangia extended terminally and sympodially on sporangiophores (FIGS. 7–8). When the thallus matured, the number of sporangia varied 3–14 and were 10.5 ± 2.5 µm (n = 61) diam. Sporangiophores were unbranched and tubular, and their average length was 42 ± 3.2 µm (n = 22) (FIGS. 7–8). A septum was not observed in the sporangiophores (FIG. 8). Nuclei usually were found within sporangia and also were observed occasionally within holdfasts and sporangiophores, although nuclei were rarely apparent within bulbous rhizoids (FIGS. 10–11).

View larger version (76K): [in this window] [in a new window]   FIGS. 10–11. The results of DAPI staining. 10. Black and white photos for contrast, bar = 5 µm. 11. Fluorescence photographs revealing numerous spherical nuclei in each sporangium and also, occasionally, in holdfasts and sporangiophores, bar = 5 µm. all of which were separated into six groupings for the two analyses (FIGS. 13–14). According to the phylogenetic trees conducted above, isolate W101 clearly was able to be grouped together with C. communis CY50

Although vegetative growth of Cyllamyces aberensis (Ozkose et al 2001) resembles that of Caecomyces sympodialis isolate W101, in having a spherical holdfast and long sporangiophores, the latter species features unbranched sporangiophores and the propensity to develop bulbous rhizoids, which would appear to be characteristically unique morphology of Caecomyces during its vegetative stage.

Carbon-source utilization.— – Isolate W101 was able to use various substrates including glucose, cellulobiose, carboxylmethyl cellulose and filter paper as carbon sources, but we could not get it to grow well on xylan (oat spelt) or rice straw. The morphological development of W101 was similar when grown on the various substrates. In addition, when isolate W101 was cultured on filter-paper strips, the strips were loose and the cellulose fragments were seen to be suspended in the broth 4–7 d (FIG. 12).

View larger version (115K): [in this window] [in a new window]   FIG. 12. Caecomyces sympodialis New W101 culture on filter paper strips Day 1 (left). After incubation 4–7 d the strips became progressively looser and the cellulose fragments suspended in the broth (right).

The maximum parsimony- and maximum evolution-based algorithms were employed in the phylogenetic analyses as mentioned above, and the relationships as performed on 22 rumen fungal partial 18S rDNA and ITS1 sequences Domains I and II are depicted (FIGS. 13–14). The groupings of these isolates for the two analyses appeared to be similar. Those isolates that were designated as belonging to the same genus when examined with respect to morphology were clustered together in a specific clade and six genera, namely Neocallimastix, Piromyces, Caecomyces, Cyllamyces, Orpinomyces and Aneromyces, (FIGS. 13–14). These results validate the conclusion that isolate W101 is phylogenetically more closely related to C. communis CY50 than it is to other genera of anaerobic rumen fungi and also support the notion that isolate W101 is a member of the genus Caecomyces.

View larger version (8K): [in this window] [in a new window]   FIGS. 13–14. Phylogenetic analyses of ITS1 sequence. Abbreviations of fungal isolates are provided (TABLE I). 13. The resultant consensus tree was produced with maximum parsimony analysis. 14. An unrooted consensus tree as generated by the use of the maximum-evolution algorithm.

KEY TO THE GENERA OF NEOCALLIMASTICACEAE AND SPECES OF THE GENUS CAECOMYCES

1. Vegetative thallus with rhizoids 2 1. Vegetative thallus without rhizoids Cyllamyces     2. Rhizoid filamentous 3     2. Rhizoid bulbous 6 Caecomyces 3. Monocentric thallus 4 3. Polycentric thallus 5     4. Uniflagellate zoospores Piromyces     4. Multiflagellate zoospores Neocallimastix 5. Polycentric thallus and uniflagellate zoospores Anaeromyces 5. Polycentric thallus and multiflagellate zoospores Orpinomyces     6. Thallus possesses unisporangiate or short sporangiphores 7     6. Thallus possesses long and unbranched sporangiphores C. sympodialis 7. Sporangium with a single bulbous rhizoid C. equi 7. Sporangium with one or more bulbous rhizoids C. communis

	ACKNOWLEDGMENTS

 
The authors thank Mr. Jin-Xian Wu and his colleagues (Livestock Research Institute, Hengchun, Taiwan) who helped us to obtain the rumen materials required for this study. The financial support of the National Science Council (Grant No. NSC93-2313-B-020-009), Taipei, Taiwan, R.O.C., is also greatly appreciated.

	FOOTNOTES

 
Accepted for publication October 3, 2006.

¹ Corresponding author. Mailing address: No. 1, Shuehfu Road, Neipu, Pingtung, Taiwan 91201. Phone: (886) 87703202 ext 5186. Fax: (886)87740550. E-mail: ox{at}mail.npust.edu.tw

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY AND DISCUSSION LITERATURE CITED

 
Brookman JL, Mennim G, Trinci AP, Theodorou MK, Tuckwell DS. 2000. Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 18S rRNA. Microbiology 146:393–403.[Abstract/Free Full Text]

Chen YC, Chien CY, Hseu RS. 2002. Piromyces polycephalus (Neocallimasticaceae), a new rumen fungus. Nova Hedwig 75:111–115.

———, Hseu RS, Cheng KJ. 2003. The genetic similarity of different generations of Neocallimastix frontalis SK. FEMS Microbiol Lett 221:227–231.[CrossRef][Medline]

Fonty G, Joblin KN, Brownlee A. 1990. Contribution of anaerobic fungi to rumen function. In: Hoshino S, Onodera RO, eds. The rumen ecosystem. Tokyo: Japan Scientific Press. p 93–100.

Gold JJ, Heath IB, Bauchop T. 1988. Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov. sp. nov., assigned to the Neocallimasticaceae. BioSystems 21:403–415.[CrossRef][Medline]

Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98.

Ho YW, Barr DJ. 1995. Classification of anaerobic gut fungi from herbivores with emphasis on rumen fungi from Malaysia. Mycologia 87:655–677.[CrossRef]

Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–63.[Abstract/Free Full Text]

Li J, Heath B, Packer L. 1992. The phylogenetic relationships of anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiomycota. I. Cladistic analysis of rRNA sequences. Can J Bot 70:1738–1740.[CrossRef]

———, ———, ———. 1993. The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiomycota. II: cladistic analysis of structural data and description of Neocallimasticales ord.nov. Can J Bot 71:393–407.

Orpin CG. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J Gen Microbiol 91:249–62.[Abstract/Free Full Text]

———. 1976. Studies on the rumen flagellate Sphaeromonas communis. J Gen Microbiol 94:270–80.[Abstract/Free Full Text]

Ozkose E, Thomas BJ, Davies DR, Griffith GW, Theodorou MK. 2001. Cyllamyces aberensis gen.nov. sp.nov., a new anaerobic gut fungus with branched sporangiophores isolated from cattle. Can J Bot 79:666–673.[CrossRef]

Retief JD. 2000. Phylogenetic analysis using PHYLIP. Methods Mol Biol 132:243–58.[Medline]

Trinci AP, Davies DR, Gull K, Lawrance MI, Theodorou MK. 1994. Anaerobic fungi in herbivorous animals. Mycol Res 98:129–152.[CrossRef]

Wubah DA, Fuller MS. 1991. Studies on Caecomyces communis: morphology and development. Mycologia 83:303–310.[CrossRef]

This article has been cited by other articles:

				A. H. Kingston-Smith, T. E. Davies, J. E. Edwards, and M. K. Theodorou From plants to animals; the role of plant cell death in ruminant herbivores J. Exp. Bot., February 4, 2008; (2008) erm326v1. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, Y.-C. Articles by Cheng, T.-Y. Search for Related Content PubMed Articles by Chen, Y.-C. Articles by Cheng, T.-Y. Agricola Articles by Chen, Y.-C. Articles by Cheng, T.-Y.