Beef cattle that respond differently to fescue toxicosis have distinct gastrointestinal tract microbiota.

Tall fescue (Lolium arundinaceum) is a widely used forage grass which shares a symbiosis with the endophytic fungus Epichloë coenophiala. The endophyte produces an alkaloid toxin that provides herbivory, heat and drought resistance to the grass, but can cause fescue toxicosis in grazing livestock. Fescue toxicosis can lead to reduced weight gain and milk yields resulting in significant losses to the livestock industry. The objective of this study was to identify bacterial and fungal communities associated with fescue toxicosis tolerance. In this trial, 149 Angus cows across two farms were continuously exposed to toxic, endophyte-infected, fescue for a total of 13 weeks. Of those 149 cows, 40 were classified into either high (HT) or low (LT) tolerance groups according to their growth performance (weight gain). 20 HT and 20 LT cattle balanced by farm were selected for amplicon sequencing to compare the fecal microbiota of the two tolerance groups. This study reveals significantly (q<0.05) different bacterial and fungal microbiota between HT and LT cattle, and indicates that fungal phylotypes may be important for an animal's response to fescue toxicosis: We found that fungal phylotypes affiliating to the Neocallimastigaceae, which are known to be important fiber-degrading fungi, were consistently more abundant in the HT cattle. Whereas fungal phylotypes related to the genus Thelebolus were more abundant in the LT cattle. This study also found more pronounced shifts in the microbiota in animals receiving higher amounts of the toxin. We identified fungal phylotypes which were consistently more abundant either in HT or LT cattle and may thus be associated with the respective animal's response to fescue toxicosis. Our results thus suggest that some fungal phylotypes might be involved in mitigating fescue toxicosis.

A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius).

Two cultures of anaerobic fungi were isolated from the forestomach of an Indian camel (Camelus dromedarius). Phylogenetic analysis using both the internal transcribed spacer (ITS) and large-subunit (LSU) regions of the rRNA locus demonstrated that these isolates were identical and formed a distinct clade within the anaerobic fungi (phylum Neocallimastigomycota). Morphological examination showed that these fungi formed monocentric thalli with filamentous rhizoids and uniflagellate zoospores, broadly similar to members of the genus Piromyces. However, distinctive morphological features were observed, notably the pinching of the cytoplasm in the sporangiophore and the formation of intercalary rhizoidal swellings. Since genetic analyses demonstrated this fungus was only distantly related to Piromyces spp. and closer to the polycentric Anaeromyces clade, we have assigned it to a new genus and species Oontomyces anksri gen. nov., sp. nov. Interrogation of the GenBank database identified several closely related ITS sequences, which were all environmental sequences obtained from camels, raising the possibility that this fungus may be specific to camelids.

The effect of anaerobic fungal inoculation on the fermentation characteristics of rice straw silages.

AIMS: To identify whether the supplement of anaerobic fungi isolates with cellulolytic activities accelerates the silage fermentation. METHODS AND RESULTS: Three fungal isolates with the highest cellulolytic activities among 45 strains of anaerobic fungal stock in our laboratory were selected and used as silage inoculants. The rice straw (RS) was ensiled for 10, 30, 60, 90 and 120 days with four treatments of anaerobic fungi derived from the control (no fungus), Piromyces M014 (isolated from the rumen of the Korean native goat), Orpinomyces R001 (isolated from the duodenum of Korean native cattle) and Neocallimastix M010 (isolated from the guts of termites), respectively. The silages inoculated with pure strains of fungi showed a higher fungal population (P < 0.05) when compared to the control silage. In situ ruminal DM disappearance of RS silage (RSS) was improved with fungal treatment. SEM observation showed live fungal cells inoculated in RS could survive during the ensiling process. Overall, this study indicated that the inoculation of anaerobic fungi decreased the cell wall content of the RSS and increased in situ dry matter disappearance. CONCLUSIONS: The supplementation of anaerobic fungi isolates to RSS as a silage inoculant improves the RSS quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing the potential application of supplement of anaerobic fungi isolated from the guts may be applied industrially as an alternate feed additive that improves the silage quality.

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture.

AIMS: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. METHODS AND RESULTS: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. CONCLUSIONS: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan-lignin complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.

Studies on carboxymethyl cellulase and xylanase activities of anaerobic fungal isolate CR4 from the bovine rumen.

An anaerobic fungal isolate, CR4, was isolated from the bovine rumen. The DNA sequence of internal transcribed spacer region 1 showed that CR4 belonged to the genus Caecocmyces. The dry matter digestibility of timothy hay by anaerobic fungal isolate CR4 was determined. The effects of carbohydrate growth substrates on carboxymethyl cellulase (CMCase) and xylanase activities also were examined. The extent of dry matter digestibility of timothy hay was 31% at 6 days' incubation. The highest specific activity of CMCase in the culture supernatant (SN) fraction was observed in xylose culture. The activity of CMCase was not detected in the SN fraction of cellobiose and xylan or in the cell-bound fraction of all growth substrates. The highest specific activity of xylanase in the SN fraction was observed in glucose culture. These results suggest that fiber-degrading enzyme activities were affected by growth substrates and that CR4 is xylanolytic. Zymogram analysis showed that CR4 produces three CMCases of molecular mass (95, 89, and 64 kDa) and three xylanases of molecular mass (82, 73, and 66 kDa). This is the first demonstration showing the molecular mass of fiber-degrading enzymes of Caecomyces.

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen.

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.

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

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.

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria.

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.

Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi.

Ribosomal ITS1 and ITS2 fragments from 8 isolates of polycentric rumen anaerobic fungi were PCR-amplified and sequenced; the sequences obtained were aligned with published data and phylogenetic analyses were performed. Analysis of the ITS1 fragment clearly differentiated between the two polycentric genera Orpinomyces and Anaeromyces and this classification is supported by morphological observation. A multi-order phylogram based on ITS2 sequences proved that anaerobic rumen fungi are separated from aerobic chytrids, which form a well-supported monophylum with the highest possible bootstrap proportion values of 100%. Sequence analysis of ITS regions is a powerful tool for classification of anaerobic fungi but morphological description of strains is still necessary because some genera of rumen fungi display a high genetic heterogeneity.

Glycoside hydrolase production by an anaerobic rumen fungus Caecomyces communis.

The ruminal fungus Caecomyces communis was grown anaerobically either in a discontinuous cultivation system or in a fermentor with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The best culture conditions for glycoside hydrolase production were obtained in Lowe medium with daily fresh medium addition, whereas the Orpin medium with ruminal fluid was favourable to fungal growth and to the enzyme export process. Among glycoside hydrolases assessed in both culture fluid and cellular homogenate, beta-D-fucosidase activity was preponderant. Most studied enzymes were mainly associated with cells (from 50% to 99%). Glycoside hydrolase activities were constitutive, but their level was regulated by a carbon source. beta-D-fucosidase and beta-D-xylosidase activity production was activated by the association of glucose plus cellobiose, whereas beta-D-glucosidase activity production was stimulated by cellobiose alone. Enzyme release could be favoured by glucose alone or by Ray grass hay added to glucose plus cellobiose.