Acid Phloroglucinol Research Articles

<b>Histochemical changes during the ontogeny of <i>malagueta</i> and <i>biquinho</i> pepper seeds

This study aimed to evaluate the structural changes and reserve deposition of malagueta and biquinho pepper seeds and to determine their chemical composition during ontogeny. The fruits were collected 25, 40, 55, 70, 85, and 100 days after anthesis (DAA). After collection, the seeds were extracted and fixed in FAA 50 and then stored in 70% ethanol. Subsequently, the seeds were embedded in methacrylate (Historesin) and sectioned with a microtome. The sections were stained with Toluidine blue O for anatomical analysis and Xylidine Ponceau, Periodic acid Schiff (PAS) and Lugol’s iodine solution for histochemical analysis. Sections were made using a cryomicrotome for acid phloroglucinol staining and Sudan III tests. Progressive lignification of the seed coat cells was observed. Proteins and lipids were the main reserve compounds of malagueta and biquinho pepper seeds stored in the embryo and endosperm. Starch served as a transient reserve during embryo development. The lipid reserves were observed in embryo and endosperm cells from 25 until 100 DAA. At 100 DAA, the protein contents of the malagueta and biquinho pepper seeds were 0.33 and 0.62 g g -1 DM and the lipid contents were 0.30 and 0.25 g g -1 DM, respectively.

Synthesis and characterization of three-arm star-shaped glassy liquid crystal containing biphenyl esters

A new three arm liquid crystalline material containing 4-Hydroxy-4`-[(p-alkoxybiphenyloxy) carbonyl] valeric acid and linked by flexible spacers tris[-4-(p-alkoxybiphenyloxy)carbonyl] valeric acid phloroglucinol ester has been synthesized and characterized by spectroscopic methods of analysis. The transition temperatures and phase behaviors were studied by Differential Scanning Calorimetry (DSC), Polarizing Optical Microscopy (POM) and X-Ray diffraction analyses. All the synthesized compounds exhibited enantiotropic nematic phase and the stability of the mesophase depends on the length of the terminal alkoxy group for the compounds studied. Keywords: Glassy phase, Liquid crystal, Nematic phase, Star shaped, Vitrification

A new flavellagic acid derivative and phloroglucinol from Rhodomyrtus tomentosa

A new flavellagic acid derivative, 3,3′,4,4′-tetra-O-methylflavellagic acid and six known compounds were isolated from the stems of Rhodomyrtus tomentosa while a new phloroglucinol, named rhodomyrtosone I, and six known compounds were isolated from the fruit. Their structures were elucidated by spectroscopic analyses as well as by comparisons with related compounds.

Plant cell wall aromatics: influence on degradation of biomass

AbstractPlant cell walls are limited in bioconversion by aromatic constituents. Chemical and structural characteristics of specific cell walls in a variety of plants have been investigated in conjunction with their biodegradability using microscopic methods. Histochemistry, ultraviolet absorption microspectrophotometry, and response of cell walls to microorga nisms and specific enzymes identifi ed significant aromatics contributing to recalcitrance. Monocotyledonous plants, such as grasses, and dicotyledonous ones, such as alfalfa, have lignin‐type compounds within the secondary walls and middle lamella of cell walls. Cell walls that are heavily lignified as shown by positive staining reactions with acid phloroglucinol and UV absorption near 280 nm appear to be the most resistant, with examples of vascular tissues in both monocots and dicots. Grasses, however, and especially warm‐season species, are rich in low molecular weight phenolic acids ester‐linked to sugars within their cell walls, occurring in both lignified and non‐lignified cell walls. In non‐lignified tissues of grasses, phenolic acids prevent microbial degradation and appear to be a major barrier to biodegradation, particularly in warm‐season grasses, such as corn, millet, and bermudagrass. Modifications in amounts, types, and linkages of cell wall aromatics, either in naturally occurring mutants or cultivars developed by plant breeding, have effectively improved the biodegradability of lignocellulose. Microbial delignification by cellulase‐less white‐rot fungi as well as pretreatment with commercial ferulic acid esterases improve biodegradation of lignocelluloses. Cellulosic bast fibers, such as those from flax stems, are mostly free of lignin and are degraded by cellulases without further pretreatment. Recovery of aromatics after pretreatment or other processing means could provide value‐added compounds and improve the economics of bioconversion. © 2008 Society of Chemical Industry and John Wiley & Sons, Ltd

Corn Fiber: Structure, Composition, and Response to Enzymes for Fermentable Sugars and Coproducts

Corn (Zea mays L.) fiber, which is the seed coat and residual endosperm left after grain processing, is a low-value residue that contains carbohydrates and aromatic compounds that could provide value-added coproducts. Treatment of corn fiber with NaOH and assessment by gas chromatography indicated a prevalence of ferulic acid, with about 90% ester-linked in the cell walls. p-coumaric acid was much lower at about 10% of the amount of ferulic acid. Histochemical reactions employing acid phloroglucinol and diazotized sulfanilic acid indicated the presence of phenolic acids in cell walls of the pericarp and aleurone layer. Various protocols were tested using milled corn fiber and pretreatment with commercial ferulic acid esterases before cellulase treatment, and dry weight loss and sugars and phenolic acids released into the filtrate were evaluated. Ferulic acid esterases effectively degraded corn fiber and released substantial amounts of ferulic acid and sugars (e.g., glucose and xylose) in the incubation medium. Light microscopy showed that ferulic acid esterase substantially disrupted the aleurone layer but caused little visible damage to the lignified pericarp cell walls. Amounts of compounds released varied with protocols, and one study with various milling methods showed that esterase pretreatment followed by cellulase released about 2.8 to 4.4 and 1.5 to 2.9 times more ferulic acid and glucose, respectively, than cellulase alone. The highest levels for one lot of corn fiber with esterase pretreatment followed by cellulase were 3.9 and 218 mg/g of ferulic acid and glucose, respectively.

Chemical Structural Investigation of the Cotton Fiber Base and Associated Seed Coat: Fourier-Transform Infrared Mapping and Histochemistry

Fourier-transform mid-infrared mapping and histochemical staining are used to reveal the location and relative importance of chemical components involved with the base of cotton fibers and their associated seed coat. These two complementary techniques are focused on the nature of the chemical components that hold cotton fibers at their bases to the seed coat and with other portions of seed coat fragments that are often found as part of the trash component of ginned cotton. Infrared results reveal waxes or long-chain alcohols adjacent to the shank of cotton fiber bases in the outer epidermal tissue in all regions of the cotton seed; uronate anions in the outer epidermis and pigment layers surrounding the bases of the fibers and strongly present in the upper palisade layer tissue of all seed regions; compounds containing carbonyl functionality, acids, and bases, at the juncture of the upper palisade and colorless layers; tannin or pretannin-type aromatic structures in the outer pigment layer and interior to the cells in the epidermal layer of all seed coat regions; and lignin-type aromatics in the "colorless" layer of all regions of the seed coat. The infrared results are complemented by staining with Oil Red O for waxes, Ruthenium Red for pectins, acid phloroglucinol for lignins, and vanillin-HCl for tannins. The results provide a better understanding of fiber-seed interactions that are important to the development of methods for improving the separation of cotton fibers from seed coats. In turn, this will help to avoid breaking fibers and pulling out seed coat fragments with the fibers during ginning.

Histochemistry and UV-microspectrometry of cell walls of untreated and ammonia-treated barley straw

Histochemical staining reactions with acid phloroglucinol and ultraviolet (UV) absorption spectra of the individual cell walls in spring barley straw (Hordeum vulgare L.) were investigated in combination with spectrometric measurements of the dioxane-water soluble lignin. Changes in lignin structure of barley straw with ammonia treatment were also investigated. Parenchyma and metaxylem vessel walls in untreated straw stained red with acid phloroglucinol and had higher absorbances around 550 nm than did epidermis and sclerenchyma cell walls, being consistent with the λmax of coniferylaldehyde. Following a reductive treatment, the lignins isolated from untreated barley straw showed an increase in UV absorbance at 280 nm and a decrease in that around 320 nm. These regions in UV and IR absorption spectra are assigned to conjugated carbonyl groups as shown by the narrowing of the IR absorption band at 1660 cm−1, and this was consistent with the staining observation of the specific tissue walls. UV microspectrometry indicated that parenchyma cell walls were much less lignified tissues than metaxylem and protoxylem vessel walls and probably epidermal cell walls. The lignins isolated from untreated and ammonia-treated straw had similar values for empirical formulae of the C9 units, phenolic hydroxyl and methoxyl group contents, and molecular weight, although the lignin of ammonia-treated straw had a slightly higher contents of nitrogen and hydrogen. The IR bands of 1730–1680 cm−1 in ammonia-treated straw lignin also disappeared. Therefore, ammonia appeared to react with the carbon atoms of the propane side-chain. Key words: Ammonia treatment, barley straw, lignin distribution, lignin structure, staining with acid phloroglucinol, ultraviolet microspectrometry

UV Absorption Microspectrophotometry and Histochemistry of Flax and Kenaf

Abstract Flax (Linum usitatissimum L.) and kenaf (Hibiscus cannabinus L.) are the sources of fibers used for textiles and other industrial applications. Both flax and kenaf produce fibers in the bast region (Fig. 1, 2) which must be separated from other tissues by retting. Although both flax and kenaf are bast fibers, their properties are vastly different. UV absorption microspectrophotometry and histochemistry elucidate their chemistry and structure related to enzymatic retting. Aromatics such as lignins are produced by plants for protection and strength, but their presence inhibits microbial degradation, which is necessary in retting. Histochemical tests indicated variations in the site and type of aromatics within these two plants (1,2). In flax, acid phloroglucinol but not chlorine-sulfite gave positive reactions occasionally in fiber cell walls in the bast. The other cell walls in the bast did not contain aromatics by these tests, although aromatics occurred in the cuticle.

Developments in application of light and scanning electron microscopy techniques for cell wall degradation studies

The results of recent technological developments in light and scanning electron microscopy closely used for research on forage cell wall degradation in ruminants, are reviewed. The indigestibility of forages by rumen microorganisms used to be ascribed mainly to an overall presence of lignin in the plant material. However, early light microscopic observations without application of histochemical staining revealed that some leaf and stem tissues were degraded completely. The early use of lignin detecting dyes, such as acid phloroglucinol or safranin, in light microscopy made it possible to discriminate between lignified undegradable and unlignified degradable plant tissues. The introduction of the scanning electron microscope enabled a further discrimination between degradable and undegradable cell wall and cell wall layers in plant tissues. As a result of continuous improvement of the techniques used in microscopy, e.g. section to slide, mirror sectioning, microspectrophotometry and cryo-ultramilling, forage indigestibility can now be attributed to the specific deposition and location of cutin/suberin or lignin layers inside the plant cell wall. These structural layers form barriers hindering access of rumen microorganisms to degradable parts of the cell wall.

Effect of Modification of the O-Methyltransferase Activity on Cell Wall Composition, Ultrastructure and Degradability of Transgenic Tobacco

The effect of O-methyltransferase (OMT) cDNA modulation on cell wall composition, ultrastructure and rumen degradability was measured on transgenic tobacco (Nicotiana tabacum). The expression of OMT cDNA in antisense orientation (AS) inhibited OMT activity by 92% whereas expression of sense constructs led to plants either co-suppressed (CS, 98% inhibition) or overexpressing OMT activity. The cell wall residues of stems were analysed for lignin content, products of nitrobenzene oxidation (NBO) and polysaccharide content. Degradability was determined by a cellulase method. Sections of stem were stained by acid phloroglucinol and Mäule reactant. Stem samples were incubated in the rumen for 8, 24 and 48 h and observed by scanning electron microscopy (SEM). Compared to controls, OMT-depleted stems showed decreased hemicellulose content but unchanged lignin content. In contrast, syringyl units decreased by 40 and 90% in AS and CS samples respectively and NBO content followed a similar trend. Dry matter cellulase degradability was significantly improved by 3·5 and 5·6 percentage units in AS and CS samples respectively. SEM showed a greater bacterial colonisation in these samples and indicated a higher rate of rumen degradability in CS tissues than in controls. Overexpressing plants had a composition and a degradability similar to that of controls. For all the plants studied, the improvements in dry matter degradability were closely linked to the syringyl to guaiacyl ratio or to the NBO content. The modifications observed in down-regulated tobacco were similar to those produced by bm3 maize mutation, but without lignin decrease. Genetic modifications should therefore be considered for improving forage digestibility.

Microbial degradation of the apical internode of Co125 and W401 maize in the rumen

The internodes of maize lines Co125 and W401, harvested five days after anthesis were cut into three fragments of equal length. In the internodes of Gramineae there is a gradient of maturity from the base upwards. Measurements of digestibility made in situ with the nylon bag method showed that the bases of the internodes were more digestible than their tops and that line Co125 was more digestible than line W401. Lignin-specific histological stains showed clear differences in staining between the sclerenchyma, fibres and xylem. The parenchyma was never stained. Lignification of tissues occurred first in the xylem and then in the fibres and sclerenchyma. There were great differences in the percentage of area stained by acid phloroglucinol, in relation to total stem area, in the bases, which were of comparable digestibility. Conversely, the percentage of area stained in the tops, which were not of comparable digestibility, differed little. SEM and TEM observations showed that the cell walls of lignified tissues were thicker in the tops than in the bases in both maize lines. They also showed that the most intensely stained tissues were the most resistant to microbial degradation. The parenchyma was degraded very rapidly but not at the same rate in all samples (base Co125 > top Co125 > base W401 > top W401) whereas the xylem was never degraded. The most extensively degraded tissues in all samples were the fibres and the parenchyma. The bases of the two maize lines were more degraded than the tops. Of the cell walls that reacted positively to lignin-specific stains only the secondary walls of the fibres and sclerenchyma were sometimes degraded. However, the sclerenchyma secondary wall was not always degraded in the top of maize W401. The phenolic compounds seemed to have different characteristics in the bases and tops of the two maize lines. This study shows the suitability of the internode as a model for observing the growth and lignification of cell walls and for determining the effects on digestibility.

Use of histochemistry, section-to-slide digestion and image analysis for evaluation of digestibility in reed canarygrass (Phalaris arundinacea L.) genotypes

A useful tool for both nutritionists and plant breeders would be the development of forage quality selection techniques that are simple, rapid and inexpensive. This study evaluates histochemistry, section-to-slide and image analysis techniques for their usefulness in a forage digestibility evaluation program. Digestibility of stem and leaf material for nine parental genotypes of the reed canarygrass (Phalaris arundinacea L.) cultivar Rival were compared using a gravimetric digestion technique. Based on differences observed for stem digestibility, two low- and two high- digestibility genotypes were selected for further study. The four genotypes were harvested for stem material at three stages of growth. Tissues examined included the epidermis (E), cortex (C), phloem (P), sclerenchyma ring (SR), xylem (X), vascular bundles (VB), and parenchyma (PA). Histochemistry staining, using acid phloroglucinol (AP) or chlorine sulphite (CS), indicated genotype and growth stage differences with all tissues except P and X. However, only the AP staining of the SR and PA, and the C with both stains were similar for the two low-digestibility genotypes. Section-to-slide digestion also indicated genotype and growth stage differences, but did not provide similar values for the two low-digestibility genotypes. Tissue area percentages, determined by image analysis, were different among genotypes for P, SR, and PA. A growth stage effect was observed for SR tissue area. The only tissues with similar area percentages for the two low-digestibility genotypes were the PA and SR. The results indicate potential use for histochemistry and image analysis to determine differences in plant stem digestibility. The SR and PA may be the key tissues involved in digestibility differences among genotypes and growth stages. Key words: Forage, digestibility, microanatomy, histochemistry, section-to-slide, image analysis

The influence of ageing on cell wall composition and degradability of three maize genotypes

Stem segments of the maize ( Zea mavs L.) hybrids LGH, Eta Ipho (EI) and a brown midrib mutant. INRA 260 bm3 (bm3) were freeze-dried, ground and analysed for cell wall content, hemicellulose, cellulose, lignin and in vitro cell wall degradability with rumen fluid. Stem cross-sections, stained with acid phloroglucinol (AP) and chlorine sulphite (CS) showed an increased intensity in staining during maturation, but no considerable difference in staining intensity was observed between genotypes. The lignin content increased during maturation with evidently less lignin in bm3 than in EI and LGH. However, cell wall degradability did not differ between the older stem segments of EI and bm3, although the amount of lignin in LGH was twice that of bm3. It can be concluded that an increase in lignin content occurs simultancously with a decrease in cell wall degradability within a genotype. However, between different genotypes the lignin content is not an indicator of degree of cell wall degradability.

Differences in rumen bacterial degradation of morphological fractions in eight cereal straws and the effect of digestion on different types of tissues and mechanical properties of straw stalks

Eight different straw species were hand-harvested and dissected into internode, node within each internode height, leaf sheath and leaf blade. Each morphological fraction was analyzed separately for cell wall constituents and in vitro digestibility. Additionally, sections of internodes were analyzed for shear stress before and after in vitro digestion, and internode cell types were evaluated for histochemical reactions for lignin and degradation by rumen microorganisms using microscopy. In vitro digestibility of cell walls (IVDCW) varied widely between species and internode heights, ranging in values as follows: internodes, 10.3–52.3%; leaf sheaths, 27.8–75.8%; leaf blades, 46.6–81.7%. Acid phloroglucinol (AP) did not stain unlignified parenchyma in straw internodes, and these tissues were totally degraded. In this respect, AP was superior to diazotized sulfanilic acid in indicating recalcitrant cell types. No relationship of IVDCW to loss of entire tissue by microbial degradation (determined by scanning electron microscopy) was found. Lignified, thick cell walls of structural tissue in internodes of some straws were markedly colonized and eroded by microorganisms and, as a result, contributed substantially to total IVDCW of this plant fraction. In internodes, the reduction of shear forces after incubation with rumen microorganisms appeared to be related to microbial erosion of structural tissue. The shear stress of internodes and compression forces to crush nodes increased from upper to lower internodes.

Relationship between structural development of cell walls and degradation of tissues in maize stems

AbstractSections of internodes of growing maize stems were used to study the behaviour of cell walls of different tissues during in‐vitro degradation with rumen fluid. Tissues with primary cell walls‐middle lamellae, at early stages of development, were degraded completely. In specific tissues, newly synthesised secondary walls were highly digestible whereas the primary walls‐middle lamellae of these tissues were indigestible. These primary walls‐middle lamellae stained positively with acid phloroglucinol but showed no fluorescence. At pollination, when secondary walls were of considerable thickness, these walls were still completely digestible even though they stained intensely with acid phloroglucinol and showed reduced fluorescence. However, at some distance from the cut end of sections, the secondary walls of the elongated tube‐like cells of sclerenchyma tissue showed considerable reduction in digestibility. Cross‐sectional area and dry weight measurements of different stem tissues revealed the importance of secondary wall digestion of sclerenchyma compared with the thin‐walled parenchyma. Chemical treatment with KmnO4 or NaOH resulted in colourless secondary walls after staining whereas primary walls still reacted positively. It was concluded that very small amounts of phenolic compounds (lignin) located in the primary wall‐middle lamella that are not removed by KmnO4 and NaOH treatment are responsible for the decrease in digestibility of tissues during plant development. Histochemical ‘lignin’ reactions and fluorescence just detect phenolic compounds and cannot be correlated with degradation.

Microscopic investigation of changes in histology and digestibility in the rumen of a forage grass and a forage legume during the first growth stage

An Italian "Dalita" ryegrass (Lolium italicum) and a European lucerne (Medicago sativa) were harvested at 5 different growth stages to determine the anatomical factors limiting their digestibility and in particular the effects of lignification of the tissues. In vitro digestibility, cell wall contents of the whole plant and stem of lucerne and of the whole plant, stem and leaf blade of ryegrass were determined. The rate and the extent of degradation in the rumen of the different tissues were observed by scanning electron microscopy. This degradation occurred very rapidly with the lucerne stems; the xylem of lucerne was the only undegradable tissue whatever the stage. The collenchyma was degraded in the rumen although with acid phloroglucinol it stained positive for the presence of phenolic compounds. Ryegrass stems were digested more slowly than lucerne stems, and the sclerenchyma and xylem of ryegrass were indigestible whatever the stage. The parenchyma located close to the sclerenchyma became indigestible as the cell walls lignified progressively from the third stage. These results contribute to the understanding of the decrease in digestibility over the first growth stage and the variation in rate of digestion of lucerne and ryegrass in the rumen.

Diazonium Compounds Localize Grass Cell Wall Phenolics: Relation to Wall Digestibility

Phenolic compounds in plant cell walls limit forage digestibility. The objective of this work was to compare three diazonium salts with more widely used lignin stains for their ability to stain phenolic compounds in grass cell walls and to relate the results to cell wall digestibility. Acid phloroglucinol, chlorine‐sulfite, azure B, diazotized sulfanilic acid, diazotized p‐nitroaniline, or p‐nitrobezenediazonium tetrafluoroborate was reacted with a series of low molecular weight phenolic compounds (acids, aldehydes, and esters), lignin extracts, and intact plant cell walls in leaf blades and stems of ‘Coastal’ bermudagrass [Cynodon dactylon (L.) Pers.]. In vitro digestibility of individual plant cell walls was assessed by microscopy of sections incubated with rumen fluid. Diazonium salts were more effective than acid phloroglucinol, chlorine‐sulfite, or azure B in forming colored compounds with phenolic acids, aldehydes, and esters. Lignin preparations from grasses gave positive reactions with all stains, and differences in color reactions occurred with some plants. Polysaccharides did not have positive histochemical reactions with the exception of larchwood xylan, which apparently contained phenolics that reacted with all stains except acid phloroglucinol and azure B. The diazonium salts and chlorine‐sulfite tended to give positive reactions with the same cell wall types, but the salts gave more stable colors and no false negatives. Treatment of a series of warm‐and cool‐season grass leaf blades with diazotized sulfanilic acid indicated positive reactions with indigestible tissues of both grass types and with the slowly or partially digested cell walls of warm‐season grasses. Diazonium compounds offer an improved method to characterize histochemically the phenolics of plant cell walls.

Microspectrophotometry of bermudagrass (Cynodon dactylon) cell walls in relation to lignification and wall biodegradability

AbstractThe walls of the major plant cell types, sclerenchyma and parenchyma, of the second (upper) and fifth (lower) internodes from the apex of bermudagrass (Cynodon dactylon L) stem have been examined by ultraviolet absorption microspectrophotometry and the results related to wall digestibility (measured in vitro with rumen liquor) and to histochemical reactions for ‘lignin’ and phenolics. Sclerenchyma walls from the lower internode gave high values of absorbance in the ultraviolet region of the spectrum equivalent to 109 mg trans‐ferulic acid g−1 dry walls; similar walls from the upper internode also gave high values but these were lower than values from the lower internode. Histochemical examination showed that the sclerenchyma walls, which were indigestible to rumen microorganisms, gave positive tests with acid phloroglucinol reagent for lignin. Parenchyma walls, which were either digested or partially digested, gave much lower absorbance values in the ultraviolet region and negative tests with acid phloroglucinol but positive tests with diazotised sulphanilic acid (upper and lower internodes) and chlorine‐sulphite (lower internode) reagents. Ultraviolet absorption microspectrophotometry is a useful technique for examining phenolics within individual cell walls that vary in biodegradability.

Influence of stage of forage maturity and ammoniation of wheat straw on ruminal degradation of wheat forage tissues

The association of ruminal microorganisms with plant tissue is important for digestion and additional information regarding specific plant/microbial interactions is needed for a more complete understanding of forage utilization. The objectives of these studies were to characterize changes in the primary anatomical structures of immature and mature wheat forage during ruminal degradation in sacco, and to evaluate microbial groups, especially rumen fungi, involved in the initial degradation of the forage. Observations were made for untreated (US) and ammoniated (AS) wheat straw. Regardless of stage of wheat forage maturity, susceptibility of leaf tissues to disappearance decreased in the following order: mesophyll and parenchyma bundle sheath > phloem > epidermis > sclerenchyma, with virtually no disappearance observed for lignified vascular tissue. Leaf blades of immature wheat forage were degraded more rapidly in sacco than those of mature forage, but similar tissues were degraded at each maturity stage. Stem material increased in content with plant age and had tissues that were highly lignified. Only small losses of tissues (primarily in the phloem, cortex and part of the parenchyma) were observed in stems. The ionophore, lasalocid, did not substantially alter the degradation patterns of immature or mature wheat forage tissues. Ruminal bacteria were the primary microorganisms responsible for the degradation of wheat forage tissues. Ruminal fungi, while not prevalent on immature leaves, were prevalent colonizers of lignified tissues in mature leaves and stems. Protozoa were not associated with wheat forage tissues during ruminal degradation in sacco. Ammoniation increased the N content of wheat straw from 5.1 to 14.7 g kg −1 dry matter (DM) and DM in vitro digestibility from 460 to 555 g kg −1 DM, and caused a slight reduction in the intensity of the acid phloroglucinol and chlorine sulfite reactions for lignin. Voluntary DM consumption by steers was 30% greater ( P < 0.01) for AS than for US. Similar lignified tissues made up the residues of US and AS after 70 h of ruminal incubation in sacco. Numbers of ruminal fungi were not affected by type of straw in the diet, but tended ( P < 0.07) to be influenced by the type of straw that was incubated in sacco in the rumen. Ruminal fungi colonized US and AS, and sporangia reached maximum numbers at 20 h and declined thereafter. The increased intake of AS was not associated with a greater number of ruminal sporangia attached to straw particles. Therefore, ruminal fungi do not appear to be involved in the mechanism by which ammoniation increases voluntary consumption of straw.

Biological structure of lignocellulose and its degradation in the rumen

Variations in lignification and digestibility occur among the lignocelulosic fibres in forages. Tissues which stain positive for lignin with acid phloroglucinol (i.e., mestome sheath and xylem in grass leaf blades; epidermis, sclerenchyma ring and xylem in grass stems) are totally inert to microbial attack and attachment for the most part. Conversely, tissues which stain for lignin with the chlorine sulphite test (i.e., sclerenchyma in grass leaves and parenchyma in grass stems) are not as resistant to attack, although these tissues are rigid and poorly degraded. Chemical treatment appears to make the chlorine sulphite fibre available for microbial attack, but not the acid phloroglucinol fibre. It is suspected that the predominant phenolic monomers in these two types of lignin differ and this difference influences the availability of the fibres. Polymeric lignin binds structural carbohydrates, rendering the sugars unavailable for microbial fermentation. In addition, low molecular weight phenolic compounds (e.g., phenolic acids) also appear to bind glucans and xylans in an undigestible complex. p-Coumaric acid seems to be especially detrimental to forage digestibility and this acid is also toxic to remen micro-organisms at lower concentrations than other acids. Brown midrib mutants in maize and sorghum are higher in digestibility than their normal counterparts and have lower lignin contents and less p-coumaric acid. In general, rumen bacteria and fungi colonize rigid plant tissue prior to degradation and their enzymes are then able to degrade the structural carbohydrates. Rumen fungi are better able to degrade leaf blade sclerenchyma than the bacteria and under specific conditions can slightly degrade xylem fibre, indicating a greater potential to break down lignocellulosic material.