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Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen

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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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slide1

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…

slide3
Ruminal anaerobic fungi, an emerging group of animal probiotics, account for upto 8% of the microbial biomass in rumen and actively colonize plant cell-walls
  • Gain access to plant materials that is unavailable to other rumen microorganisms

contd…

slide4
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
  • 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)
slide6
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)

slide7
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
  • 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).
slide9
Division: 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
  • 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)
contd
contd…
  • 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
  • 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)
slide15

contd…

  • 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
  • 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
  • Genus identification:
    • Number of flagella per zoospore
    • Rhizomycelium
    • Shape of sporangium
  • (Breton et al, 1990; Asao et al, 1993)

contd…

slide18
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
  • 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
  • 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)

slide21

contd…

  • 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)

slide22

contd…

  • 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)

slide23

contd…

  • 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)

slide24
Fungi degrades plant 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
  • 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…
  • 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…
  • 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
  • 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)

slide29

contd…

  • 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

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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