A scanning electron microscopy study of the invasion of leaflets of a bloat-safe and a bloat-causing legume by rumen microorganisms

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Abstract
A newly developed technique using ruthenium red to detect foci of bacterial digestion in mounts of whole leaflets that had been incubated with rumen bacteria was used to compare the digestion of alfalfa, a bloat-causing legume, and sainfoin, a bloat-safe legume. When whole leaflets were suspended in an artificial rumen medium and inoculated with rumen bacteria, massive bacterial adhesion and proliferation were noted at the stomata of alfalfa leaflets after 6 h of incubation, whereas only a few isolated bacteria adhered near the stomata of sainfoin leaflets. After 22 h of incubation, the epidermal layers of alfalfa leaflets had peeled away in many areas, revealing an extensive bacterial invasion of the underlying mesophyll tissue in which large bacterial microcolonies had formed in intercellular spaces, and in intracellular spaces in several areas where plant cell walls had broken down. After 22 h of incubation, the surface of sainfoin leaflets resembled that of alfalfa leaflets at 6 h, with bacterial microcolonies adhering to the area surrounding the stomata, but without sloughing of the epidermis. Uninoculated control leaflets of both species showed no surface alterations but part of their normal bacterial flora had proliferated to form microcolonies on the surface after 22 h of incubation.Dry matter loss due to leaching or bacterial digestion when whole leaflets of legumes were suspended in an artificial rumen medium, alone or with rumen bacteria, was significantly higher in the bloat-causing group. Values of leaching and of bacterial digestion were positively correlated.We conclude that reported differences in plant anatomy, and in cell wall chemistry, produce distinct rates of organic nutrient release from legume leaflets, and that these same differences produce an equally distinct susceptibility of leaflets to bacterial invasion, plant cell rupture, and the consequent release of intracellular plant components. The rate of release of organic nutrients from legume leaflets may be important in the etiology of foamy pasture bloat.This technique of in vitro digestion of whole leaflets followed by ruthenium red staining shows some promise of providing a rapid and qualitative test to distinguish, within a species, cultivars that may differ in their bloat-related characteristics.