Sequence of Events in the Digestion of Fresh Legume Leaves by Rumen Bacteria

Abstract

When fresh whole leaves of six different species of forage legumes were suspended in an artificial rumen medium and inoculated with rumen bacteria, bacterial adhesion and proliferation were noted at the stomata, and penetration of the stomata by these bacteria was documented by electron microscopy. The invading bacteria adhered to surfaces within the intercellular space of the leaf and produced very extensive exopolysaccharide-enclosed microcolonies. After some of the legume leaf cell walls were disorganized and ruptured by bacterial digestion, these cells (notably, parenchyma and epidermal cells) were invaded by bacteria, with subsequent formation of intracellular microcolonies. However, other cells were neither ruptured nor colonized (notably, stomata guard cells and vascular tissue). At all stages of the digestion of intact legume leaves, the rumen bacteria grew in microcolonies composed of cells of single or mixed morphological types, and a particular ecological niche was often completely and consistently occupied by a very large microcolony of cells of single or mixed morphological types.The bacterial digestion of cut grass leaves and of cell walls prepared from various grasses has been documented by several groups (1,2,9,13,14). Brazle and Harbers (3) studied the digestion of air-dried alfalfa hay by scanning electron microscopy, but no detailed studies of the sequence of events in the bacterial digestion of fresh whole legume leaves have been reported.Various bacteria have been shown to adhere to plant cell walls to produce "pits" by their cellulolytic activity (1, 2, 5). The cell walls of different plant tissues have shown sharp differences in the extent to which they are colonized and digested by bacteria (1, 2, 5), and workers in the United Kingdom (9,13,14) and in the southern United States (1, 2) have found that morphologically different cellulolytic bacteria predominate in the digestion of plant materials in their particular geographic regions. Studies of the adhesion of groups of rumen bacteria to their polymeric substrates have shown that amylase producers adhere to starch (11) but not to cellulose and that cellulose decomposers adhere to cellulose (17, 18) but not to starch. Thus, we expect that plant material will be heavily colonized by various types of bacteria soon after it is introduced into the rumen; therefore, it is not surprising that Forsberg and Lam (10) found 75% of the adenosine triphosphate of the bacteria in the rumen contents to be associated with food particles.Because transmission electron microscopy of ruthenium red-stained sections allows both bacteria and their exopolysaccharide products to be seen throughout leaf tissues during digestion, we incubated intact legume leaves with rumen bacteria and examined them at intervals using this method. We have shown (7) that the rumen bacterial population is made up of three distinct subpopulations-the rumen fluid bacteria, the food particle-associated bacteria, and the bacteria adherent to the rumen epithelium. To obtain a good represen...

Successional colonization of perennial ryegrass by rumen bacteria

“…Latham et al (1978) illustrated preferential colonization of fresh PRG cut ends, but as our leaf blades had to be cut for mounting onto the stubs, we were unable to see degree of colonization of cut ends. Cheng et al (1980), on the other hand, illustrated that rumen bacteria commonly colonized around the stomata of various fresh forages and postulated that this was the main mechanism of bacterial plant invasion. However, we found little evidence that rumen bacteria preferentially…”

“…colonized stomata in these experiments. This is possibly due to the fact that in the experiments conducted by Cheng et al (1980) the plant material was not cut; thus, the easiest route of entry may well be through stomata in this instance.…”

“…Rumen microorganisms rapidly associate with and colonize recently ingested feed particles (Cheng et al 1980;Miron et al 2001;Russell and Rychlik 2001;Koike et al 2003;Edwards et al 2007Edwards et al , 2008. As the attachment of rumen microbiota to ingested forage is central for utilization of plant nutrients (McAllister et al 1994;Dewhurst et al 1996;Kingston-Smith et al 2010), understanding plant-microbe interactions is paramount in improving ruminant nutrient use efficiency.…”

Nutrient availability during the early stages of colonization of fresh forage by rumen micro‐organisms

“…5a,b) indicate that contamination of introduced plant material with typically anaerobic, ruminal microorganisms was negligible at all stages of incubation. In addition, it has been shown previously that bacteria adhering to the surface of leaves do not penetrate the intercellular spaces during 22 h anaerobic incubation in the absence of a rumen inoculum (Cheng et al, 1980). It is possible that carbohydrate recovery in the buffer failed to reach 100% of tissue carbohydrate (even after extensive incubation) because of the plants' own cellular demands for readily fermentable carbohydrate to support anaerobic fermentation (Albrecht et al, 1997;Vartapetian & Jackson, 1997).…”

Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen

“…Ruminal degradation of fresh forage differs from that of conserved 'dead' plant material (Kingston-Smith & Theodorou, 2000;Joblin et al, 2002), with very little known about how these differences affect the microorganisms involved in forage degradation (Cheng et al, 1980(Cheng et al, , 1981. As uncultivated novel taxa comprise a significant proportion of the bacterial species in feed-associated ruminal biofilms (Tajima et al, 1999;Koike et al, 2003b;Larue et al, 2005) and associated with plant material (Marilley & Aragno, 1999;Sessitsch et al, 2002;Dent et al, 2004;Jiao et al, 2006) it is clear that the use of noncultivation-based techniques is important for the study of ruminal bacteria colonizing fresh forage.…”