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Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen . __________________________ Ravinder Nagpal Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana) India. Introduction.

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Ruminal anaerobic fungi:

The potential plant-fiber degraders in the rumen

__________________________

Ravinder Nagpal

Dairy Microbiology Division,

National Dairy Research Institute,

Karnal 132001 (Haryana) India.


Introduction
Introduction

  • Importance of ruminants to mankind has led to a great deal of research into the digestion of plant fibers in order to improve the production efficiency.

  • Majority of livestock subsist on poor quality fibrous crop residues and agro-industrial wastes

  • Attempts are being made to enhance the digestibility of poor quality feeds by various feed additives

contd…


contd…


  • Rhizoids of vegetative thalli penetrate plant tissues better than bacteria and protozoa

  • Help in access of other rumen microbes to the secondary cell wall of feed particles

  • More rapid and complete degradation of forage entering the rumen through proficient fibre degrading enzymes

  • Development of direct-fed microbials (DFM) for improved rumen performance appears to be a pre-requisite for the sustainable animal production.


Rumen anaerobic fungi
Rumen anaerobic fungi than bacteria and protozoa

  • Observed in the rumen as early as 1910, but were believed to be flagellate protozoa

    (Liebetanz et al, 1910; Braune et al, 1913)

  • Confirmed to be a true fungus by the presence of chitin in its cell wall and designated as Neocallimastix frontalis

    (Orpin, 1975)

  • Identified anaerobic fungi in sheep rumen, each of which had a motile stage (the zoospore) and a non-motile zoosporangium(Orpin, 1975)


  • Found in the rumen and other parts of the gastro-intestinal tract of herbivorous animals

    (Williams et al, 1987; Rezaeian et al, 2004)

  • Play an active and positive role in fibre degradation as evidenced by the presence of different hydrolytic enzymes

    (Williams et al, 1987; Samanta et al, 2001; Paul et al, 2003)

  • There is a significant reduction in in-vitro gas production and degradation of fibrous feeds, indicating a positive role played by fungi in fibre degradation

    (Kamra et al, 2004; Lee et al, 2004; Dey et al, 2004)


  • The fibre-based diets stimulate the fungal growth in the rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

    (Kamra et al, 2003)

  • These fungi prefer to get attached to the most lignified tissues of plant feed material by producing variety of enzymes(Akin et al, 1987)


Classification
Classification rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • Based on ultrastructural characteristics of the zoospores, anaerobic fungi were assigned to the order of Spizellomycetales and in the family, Neocallimasticaceae

    (Barr et al, 1988)

  • Suggested the subdivision of this family into three genera containing monocentric species, Neocallimastix, Piromyces (previously Piromonas) and Caecomyces (previously Sphaeromonas)

    (Gold et al, 1988)

  • Three polycentric genera have been described, Orpinomyces(Barr et al, 1989), Anaeromyces(Breton et al, 1990)and Cyllamyces(Ozkose et al, 2001).


Division: rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates Eumycota

Subdivision: Mastigomycotina

Class: Chytridiomycetes

Order: Spizellomycetales

Family: Neocallimasticaceae

Genera:Monocentric:

Caecomyces:zoospores with one or two flagella; thallus with a globular rhizoid

Neocallimastix:zoospore with four to twenty flagella; thallus with filamentous branching rhizoids

Piromyces:zoospore with one to four flagella and thallus with filamentous branching rhizoids

Polycentric:

Orpinomyces:multiflagellate zoospore

Anaeromyces:zoospore with one flagellum

Cyllamyces:zoospore with one to two flagella with thalloid branched sporangiophore


Life cycle
LIFE CYCLE rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • Life cycle lasts about 23-32 hours(Joblin 1981; Bauchop 1983; Lowe et al., 1987)

  • The life cycle of monocentric fungi consists of an alteration between a motile, zoosporic stage and a vegetative, zoosporangial stage

  • Flagellate zoospores are released from a sporangium and encyst by shedding their flagella

  • The cyst germinates to produce a germ tube, which later develops into rhizoids(Orpin et al, 1977)


Teunissen and Op den Camp, 1993; Harhangi, 2002 rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates


Contd
contd… rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • The development of zoospores from young sporangia may occur within 8 hours after encystment under appropriate conditions (Orpin et al, 1977)

  • Polycentric fungi have indeterminate life cycles and are not dependent upon the formation of zoospores for their continued survival

    (Ho & Bauchop, 1991)

  • Zoospores are produced infrequently or zoosporogenesis is even absent(Phillip et al, 1989)


Distribution
Distribution rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • First isolation in the UK from the rumen of sheep(Orpin, 1975)

  • Have been found on almost all the continents and in all of the geographic regions, where there have been sought

  • Ubiquitous among ruminants such as cattle, buffalo, goat(Singhal et al, 2000; Dey et al, 2004; Thareja et al, 2006)


contd… rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • Red deer and impala

    (Bauchop et al, 1979; Singhal et al, 2000)

  • Grey kangaroo, wallaroo and swamp wallaby(Breton et al, 1989)

  • Fecal samples of hindgut fermenters such as ass, horse, elephant and zebra

    (Breton et al, 1990; Li et al, 1990)

  • Isolated from fecal and rumen samples of wild Neelgai(Paul et al, 2004; Tripathi et al, 2007)


Isolation
Isolation rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • Overlayering with partially molten agar with filtered rumen fluid(Orpin, 1975)

  • Plate culture technique for anaerobic fungi from rumen digesta of sheep and cattle (Lowe et al, 1985)

  • Roll-bottle method involving inoculating a dilution series of molten agar medium with filtered rumen fluid (Joblin, 1981)

    Penicillin, Streptomycin, Neomycin and Chloramphenicol are added to the

    isolation media to suppress the bacterial growth


Identification
Identification rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates

  • Genus identification:

    • Number of flagella per zoospore

    • Rhizomycelium

    • Shape of sporangium

  • (Breton et al, 1990; Asao et al, 1993)

contd…


  • Species are delimited on the basis of zoospore ultrastructure

    (Ho & Barr, 1995)

  • 18 species in six genera have been classified in the literature

  • Species identification by PCR-amplification and sequencing of ITS1 and ITS2

    (Brookman et al, 2000; Fliegerova et al, 2004)


Enumeration
Enumeration ultrastructure

  • Counts of individual zoospores and zoosporangia have been used to estimate fungal populations in vitro and in vivo

    (Joblin, 1981; Ushida et al, 1989)

  • Used colony-forming units per gram dry weight of feces as the basis for quantifying species of Piromyces.

    (Breton et al, 1991)

  • Procedure based on the technique of most probable numbers, was developed to enumerate rumen fungi as thallus-forming units

    (Theodorou et al, 1990)


Role of anaerobic fungi in fibre digestion
Role of anaerobic fungi in fibre digestion ultrastructure

  • Role of rumen fungi in the degradation of plant fibre has been examined extensively

    (Lee et al, 2000; 2004; Samanta et al, 2001; Dey et al, 2004, Paul et al, 2004; Thareja et al, 2006; Tripathy et al, 2007)

  • These fungi are better at penetrating plant tissue than are bacteria and protozoa(Orpin and Joblin, 1988)

  • Such penetration leads to faster and more complete degradation of forage that enters the rumen.

    (Bauchop and Mountfort, 1981)


contd… ultrastructure

  • Degradation of lignin-containing walls of plant cells is an important characteristic of rumen fungi (Mountfort et al, 1982; Akin and Benner, 1988)

  • Rumen fungi dissolve small amounts of phenolic compounds from plant cell walls(Orpin, 1983; Gordon et al, 1985)

  • Zoospores of many species appear to colonize the lignin-containing tissues preferentially and to establish colonies localized on sclerenchyma and xylem cells.

    (Akin et al, 1986)


contd… ultrastructure

  • Anaerobic fungi penetrate the cuticle, a barrier that other microorganisms cannot cross.

  • Rumen fungi attack recalcitrant plant cell walls by weakening the textural strength of the residue(Akin et al, 1989, 1990)

  • The greater ability of rumen fungi to weaken forage fibre may be important in enhancing forage utilization by the host animal

    (Borneman and Akin, 1990)


contd… ultrastructure

  • Increased digestibility of straw with use of different anaerobic fungi viz., Orpinomyces, Piromyces and Anaeromyces was observed(Manikumar et al, 2002; Sehgal et al, 2002; Tripathy et al, 2007)

  • 7-12% increase in voluntary intake of straw based diet was reported when the sheeps were dosed with cultures of monocentric fungi(Gordon and Phillips, 1998)

  • Fungal culture increased Cellulose degradation by 26% under in vitro environment.

    (Lee et al, 2004)


  • Fungi degrades ultrastructureplant cell wall:

    • Xylem and mestome bundle sheath in leaves

    • Schlerenchyma ring in stem

    • Cuticular barrier of leaves

      (Bauchop et al., 1989)


Hydrolytic enzymes
Hydrolytic Enzymes ultrastructure

  • While rumen protozoa and bacteria have been shown to play a role in plant fibre degradation

    (Williams, 1988; Akin and Benner, 1988)

  • Rumen fungi display a somewhat greater potential for the degradation of more heavily lignified plant tissues

    (Akin et al, 1988)

  • To degrade and utilize plant cell walls, anaerobic fungi produce a wide range of hydrolytic enzymes including:


Contd1
contd… ultrastructure

  • Cellulases

    (Barichievich and Calza, 1990; Yanke et al, 1993; Paul et al, 2004)

  • Hemicellulases

    (Lowe et al, 1987; Mountfort and Asher, 1989)

  • Proteases

    (Wallace and Joblin, 1985; Michel et al, 1993)

  • Amylases, Amyloglycosidases

    (Mountfort and Asher, 1988; Paul et al, 2004)


Contd2
contd… ultrastructure

  • Feruloyl and p-coumaryl esterases

    (Borneman et al, 1990; 1991; 1992; Paul et al, 2004)

  • Various disaccharidases

    (Hebraud and Fevre, 1988; Chen et al, 1994)

  • pectinases

    (Gordon and Phillips, 1992)

  • Exonucleases or avicelases

    (Cabe, 1998)


Interaction with other rumen microorganisms
Interaction with other rumen microorganisms ultrastructure

  • Anaerobic fungi form quite stable cocultures with rumen methanogenic bacteria as a result of their high production of hydrogen

    (Fonty and Joblin, 1991; Orpin and Joblin, 1997)

  • These cocultures produce an increased amount of fungal biomass and exhibit an increase in both the rate and extent of cellulose degradation

    (Bernalier et al, 1989; 1991; Joblin et al, 1989)

  • Cellulolytic activity appeared to be inhibited, when combined in coculture with the cellulolytic ruminococci

    (Bernalier et al, 1992; Roger et al, 1993)


contd… ultrastructure

  • Growth of the rumen fungi was found to be markedly inhibited in cocultures with rumen bacteria

    (Dehority and Tirabasso, 2000)

  • Coincubation of protozoa with fungi has shown that the protozoa are able to both ingest and digest fungi

    (Orpin and Joblin, 1997)

  • Chitinase activity in samples of mixed rumen protozoa account for their predatory activity on the rumen fungi

    (Joblin, 1990; Williams et al, 1994; Morgavi et al, (1994)


Potential benefits of ruminal anaerobic fungi for improved animal nutrition and productivity
Potential benefits of ruminal anaerobic fungi for improved animal nutrition and productivity

  • Improved fibre digestion and nutrient utilization

  • More feed intake and feed efficiency

  • Increased body weight

  • Improved milk production


Prospective applications of ruminal anaerobic fungi
Prospective applications of ruminal anaerobic fungi animal nutrition and productivity

Could be exploited as:

  • Direct-fed microbials

  • Animal feed additives

  • Novel silage inoculants

  • For large scale production of enzymes (e.g. cellulase)


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