Scott A. Dalzell

A rapid method for the measurement of Leucaena spp proanthocyanidins by the Proanthocyanidin (Butanol/HCl) assay

Proanthocyanidin (PA) extraction, sample preparation and proanthocyanidin assay (butanol/HCl) reaction conditions were evaluated for measuring PA in Leucaena spp leaf material. The optimal conditions for extracting PA from leaf tissue are described, with short sequential sonications in 70% aqueous acetone being as efficient as prolonged sequential mechanical agitation. In methanol–based extracts, after back extraction to remove pigments, increasing the water content of the reagent/sample matrix suppressed colour development. The addition of low concentrations of Fe3+ to the butanol/HCl reagent enhanced colour yield, but higher Fe3+ concentrations suppressed colour development. The presence of ascorbic acid in the sample extract was shown to increase colour development. Varying the reagent: sample extract ratio from 4:1 to 6:1 significantly decreased colour yield, but neither ratio was different from 5:1. Optimum conditions for the PA assay were as follows: a water content of 8%, the omission of Fe3+, a reagent: sample extract ratio of 5:1 and the addition of ascorbic acid to the stock PA standard solution to match that contributed by the extract in the final mixture. Sample preparation procedures, using back extraction to remove pigments and non-PA phenolics with diethyl ether and ethyl acetate, respectively, were time-consuming and subject to PA losses. The measurement of PA directly in the 70% aqueous acetone extract eliminated these PA losses, but the PA assay required additional optimisation for direct analysis of crude acetone extracts. In the final optimised procedure, PA was extracted by sequential sonication with 70% aqueous acetone containing 5·26 mM sodium metabisulphite as the antioxidant. These extracts were directly analysed by the butanol/HCl reaction using a reagent: sample extract ratio of 5:1, the omission of Fe3+ from the butanol/HCl reagent and the addition of sodium metabisulphite to match that contributed by the extract. This produced consistent linear calibration curves over the range 25–1000 μg PA with an average recovery of 101%.

Prevalence of mimosine and DHP toxicity in cattle grazing Leucaena leucocephala pastures in Queensland, Australia

A postal survey of the level of awareness of leucaena toxicity and an on-farm study of the toxicity status of Queensland cattle herds grazing leucaena were conducted to investigate the prevalence of mimosine and dihydroxypyridine (DHP) toxicity in Queensland. In total, 195 of 356 graziers surveyed responded to the postal survey. Sixty-three percent had inoculated their cattle with in vitro Synergistes jonesii inoculum (produced in an anaerobic fermenter) and 30% of these had inoculated more than once. The remainder used inappropriate procedures. Many graziers (43%) had occasionally observed toxicity symptoms of hair loss and poor animal growth rates. In the on-farm study, the toxicity status of 385 animals in 44 individually managed herds on 36 properties was determined by urine analysis of mimosine and DHP concentrations. No animals were experiencing mimosine toxicity, based on low concentrations of this compound found in the urine. Using the criterion that average herd urine concentrations of DHP >100 μg/mL was indicative of subclinical toxicity, 48% of herds were exposed to subclinical toxicity due to dominant 3,4-DHP (21%) or dominant 2,3-DHP (27%) toxicity; many of these herds had been inoculated with S. jonesii and were thought to be protected. The finding that 27% of herds were excreting high concentrations of 2,3-DHP was unexpected. Statistical analysis of herd-management data revealed that the method used by graziers to inoculate their herds was significantly (P < 0.05) but weakly linked to herd protection status. It was concluded that subclinical 3,4-DHP and 2,3-DHP toxicity remains a problem in Queensland and is likely to be limiting animal production in a significant number of cattle grazing leucaena-grass pastures.

A grazier survey of the long-term productivity of leucaena (Leucaena leucocephala)-grass pastures in Queensland

Leucaena leucocephala subsp. glabrata (leucaena)-grass pastures are productive, perennial and long-lived (40 years). However, little is known about changes in the productivity of these pastures as they age even though they are grazed intensively and are rarely fertilised. A postal survey of beef cattle producers in Queensland who grow leucaena pastures was conducted. The questionnaire gathered information regarding: property location; extent and age of leucaena pastures; soil type; leucaena and grass establishment methodology; grazing and fertiliser management; and grazier perceptions of changes over time in leucaena productivity, grass growth and ground cover, prevalence of undesirable grasses and weeds, and livestock productivity. Graziers were asked to report on both young (≤10 years old) and aging (10 years old) pastures under their management. Eighty-eight graziers responded describing 124 leucaena paddocks covering 11750 ha. The survey results described the typical physical and management characteristics of leucaena pastures in Queensland. Graziers reported a decline in leucaena productivity in 58% of aging pastures, and declines in grass growth (32%) and livestock productivity (42%) associated with declining leucaena growth. Leucaena decline was greater in soil types of marginal initial fertility, particularly brigalow clay, soft wood scrub, downs and duplex soils. Maintenance fertiliser was not applied to most (98%) leucaena pastures surveyed despite significant amounts of nutrient removal, particularly phosphorus and sulphur, occurring over prolonged periods of moderate to high grazing pressure. It is predicted that large areas of leucaena pasture will continue to suffer soil nutrient depletion under current management practices. Research is needed to develop ameliorative actions to reinvigorate pasture productivity.

Genotypic variation in proanthocyanidin status in the Leucaena genus

The proanthocyanidin (PA) status of 116 accessions from the Leucaena genus representing 21 species, 6 subspecies, 3 varieties and 4 interspecific hybrids was evaluated under uniform environmental and experimental conditions at Redland Bay, Queensland, Australia in October 1997. The PA content of lyophilized youngest fully expanded leaves was measured spectrophotometrically by the butanol/HCl assay referenced to L. leucocephala ssp. glabrata standard PA and expressed as L. leucocephala ssp. glabrata PA equivalents (LLPAE). Considerable interspecific variation in PA concentration existed within the genus, ranging from 0-339 g LLPAE/kg dry matter (DM). Taxa including L. confertiflora, L. cuspidata, L. esculenta and L. greggii contained very high (> 180 g LLPAE/kg DM) PA concentrations. Similarly, many agronomically superior accessions from L. diversifolia, L. pallida and L. trichandra contained extremely high (up to 250 g LLPAE/kg DM) PA concentrations, although these taxa exhibited wide intraspecific variation in PA content offering the potential to select accessions with lower (120-160 g LLPAE/kg DM) PA content. Commercial cultivars of L. leucocephala ssp. glabrata, known to produce forage of superior quality, contained low amounts of PA (33-39 g LLPAE/kg DM). Artificial interspecific hybrids had PA contents intermediate to those of both parents, Lesser-known taxa. including L. collinsii, L. lanceolata, L. lempirana, L. macrophylla, L. magnifica, L. multicapitula, L. salvadorensis and L. trichodes, contained undetectable to low (0-36 g LLPAE/kg DM) quantities of PA and have potential as parents to breed interspecific hybrids of low PA status and superior forage quality. Extractable PA was the dominant PA component, accounting for 91% of total PA within the genus. Regression analysis of accession ranks from different experiments compared to these results indicated that genetic regulation of Leucaena spp. PA content was consistent (P < 0.01) under different edapho-climatic environments. The distribution of PA within the Leucaena genus did not concur with the predictions of various evolutionary and phylogenetic plant defence theories.

Soil organic carbon and total nitrogen under Leucaena leucocephala pastures in Queensland

Soil organic carbon (OC) and total nitrogen (TN) accumulation in the top 00.15m of leucaenagrass pastures were compared with native pastures and with continuously cropped land. OC and TN levels were highest under long-term leucaenagrass pasture (P0.05). For leucaenagrass pastures that had been established for 20, 31, and 38 years, OC accumulated at rates that exceeded those of the adjacent native grass pasture by 267, 140, and 79kg/ha.year, respectively, while TN accumulated at rates that exceeded those of the native grass pastures by 16.7, 10.8, and 14.0kg/ha.year, respectively. At a site where 14-year-old leucaenagrass pasture was adjacent to continuously cropped land, there were benefits in OC accumulation of 762kg/ha.year and in TN accumulation of 61.9kg/ha.year associated with the establishment of leucaenagrass pastures. Similar C:N ratios (range 12.714.5) of soil OC in leucaena and grass-only pastures indicated that plant-available N limited soil OC accumulation in pure grass swards. Higher OC accumulation occurred near leucaena hedgerows than in the middle of the inter-row in most leucaenagrass pastures. Rates of C sequestration were compared with simple models of greenhouse gas (GHG) emissions from the grazed pastures. The amount of carbon dioxide equivalent (CO 2-e) accumulated in additional topsoil OC of leucaenagrass pastures ≤20 years old offset estimates of the amount of CO2-e emitted in methane and nitrous oxide from beef cattle grazing these pastures, thus giving positive GHG balances. Less productive, aging leucaena pastures 20 years old had negative GHG balances; lower additional topsoil OC accumulation rates compared with native grass pastures failed to offset animal emissions.

Efficacy, persistence and presence of Synergistes jonesii in cattle grazing leucaena in Queensland: on-farm observations pre- and post-inoculation

A study of eight commercial cattle herds grazing leucaena (Leucaena leucocephala subsp. glabrata) pastures was undertaken to determine (1) the efficacy of in vitro Synergistes jonesii inoculum (produced in an anaerobic fermenter) in degrading the dihydroxypyridone (DHP) isomers produced during digestion of leucaena forage; and (2) the persistence of the inoculum in the rumen of cattle following a period grazing non-leucaena pastures. Cattle were introduced to the leucaena pastures for an initial period varying from 17 to 71 days. Fourteen to fifteen animals were then sampled for (1) urine and blood plasma to determine toxicity status as indicated by concentration of DHP; (2) faeces for estimation of diet composition; and (3) rumen fluid for detection of S. jonesii by nested polymerase PCR analysis. After a further 42-56 days, animals were resampled as before to confirm toxicity status and inoculated with the in vitro S. jonesii inoculum; the herds were then sampled a third time (42-60 days after inoculation) to test the effectiveness of the inoculum in degrading DHP. Five of the herds were then removed from leucaena pastures for periods ranging from 80 to 120 days and returned to leucaena pastures for 21 days to check persistence of the inoculum as indicated by retention of capacity to degrade DHP. The data indicated (1) a very slow build up of capacity to degrade DHP isomers on some properties before inoculation; (2) frequent occurrence of high levels of 2,3-DHP in urine indicating partial toxin degradation, both before and after inoculation; (3) a low incidence of detection of S. jonesii in rumen fluid after inoculation based on nested PCR analysis; (4) failure of inoculation to degrade DHP on one of two properties tested; and (5) loss of capacity to degrade DHP on some properties after <4 months on alternative non-leucaena pastures. It was concluded that while most herds showed some capability to degrade DHP due to some residual capability from previous exposure, they did not achieve the same rapid and complete DHP degradation reported in the 1980s. Nevertheless, it was concluded that the in vitro inoculum was at least partially effective and should continue to be used by graziers until improved sources of inoculum and/or inoculation methodologies are demonstrated.

Response of Leucaena leucocephala pastures to phosphorus and sulfur application in Queensland

A series of fertiliser trials were conducted on leucaena (Leucaena leucocephala subsp. glabrata) pastures growing on a range of soil types in south-east and central Queensland. The primary objective was to determine the extent of phosphorus (P) and sulfur (S) deficiencies in leucaena-grass pastures established on either virgin soils or previously cropped soils. Two experiments were conducted across nine sites and confirmed that, for many soils in Queensland, leucaena growth was restricted by P and S nutrient deficiencies, which limited plant growth directly and suppressed symbiotic N2 fixation. The major factors contributing to the P and S deficiencies and thus affecting leucaena response were: (i) inherent low soil fertility, (ii) nutrient removal by cropping and grazing, (iii) shallow soils, (iv) soil acidity, and (v) grass competition for available water and nutrients. A secondary treatment, inter-row cultivation, had little effect on leucaena growth but significantly increased grass growth in some soils. In all these experiments, leaf S concentrations and N : S ratios in index tissue were inconsistent indicators of adequacy of S. Similarly, leaf P concentrations were not useful indicators of P deficiency due to inappropriate (drought) leaf sampling conditions experienced in these experiments. The experiments demonstrate that the productivity of leucaena-grass pastures, especially in older leucaena plantations, will be limited by nutrient deficiencies on many soils in Queensland. While leucaena yield was suppressed, no foliar symptoms of nutrient deficiency were observed. Growers need to monitor the nutrient status of their leucaena-grass pastures by leaf tissue analysis using a new sampling protocol. Strategic fertiliser application has the potential to increase rainfall use efficiency by 50% with an expected parallel increase in cattle liveweight gain.

Methods of field preservation and selection of sample tissue for condensed tannin analysis in Leucaena species

Field sample preservation and tissue selection for condensed tannin (CT) analysis were investigated with three Leucaena genotypes, L. leucocephala subspecies glabrata (Lam.) de Wit cv. Tarramba (K636), L pallida Britton and Rose (K806) and the F1 interspecific hybrid L. pallida (K748) × L. leucocephala (K636). Shoot, youngest fully expanded leaf (YFEL) and mature leaf (ML) tissues were analysed for extractable, protein and fibre-bound CT for each Leucaena species. There was no significant difference in CT content measured in excised tissues whether frozen by dry ice or liquid nitrogen. The shoot material of all genotypes contained more CT, of all components, than the YFEL and ML tissues. Extractable CT was highest in L. pallida, lowest in L. leucocephala and intermediate in the hybrid. The YFEL was selected as the most appropriate sample tissue because it was easily recognized, enabled consistent sampling of tissue at the same physiological stage of maturity, and contained sufficient CT for the determination of the CT status of each genotype.

Survey of long-term productivity and nutritional status of Leucaena leucocephala-grass pastures in subtropical Queensland

A survey of the productivity and nutritional status of leucaena (Leucaena leucocephala subsp. glabrata) pastures of different ages was conducted in subtropical Queensland from 2006 to 2008. Four leucaena stands (aged 8, 20, 31 and 38 years) growing on the same Vertosol soil type at Brian Pastures Research Station were surveyed. In the higher rainfall season of 2007-2008, leucaena yields and rainfall-use efficiency were highest in 8-year-old stands [2128 kg total dry matter (DM)/ha or 4.0 kg DM/ha.mm] and lowest in 38-year-old stands (978 kg total DM/ha or 1.9 kg DM/ha.mm). The reduced productivity in the 38-year-old leucaena pasture was associated with a decline in stem number/plant and leucaena plant density due to observed plant mortality. The reduced yield and vigour of aging leucaena was associated with nitrogen deficiency related to declining phosphorus and sulfur availability for adequate symbiotic N2 fixation and leucaena plant growth. Nutrient deficiencies of phosphorus and sulfur in leaf tissue were related to low to medium initial soil fertility (7-27 mg/kg of bicarbonate extractable phosphorus in the top 20 cm), coupled with inherent subsoil constraints (shallow soils, sodicity and high pH), and exacerbated by both long-term removal of nutrients by grazing animals and a reduction in soil phosphorus and sulfur availability over time. To maintain the productivity of leucaena pastures, plant nutritional status needs to be monitored in order to determine strategic fertiliser application.

Effect of environment and plant phenology on prediction of plant nutrient deficiency using leaf analysis in Leucaena leucocephala

Plant analysis is an important tool for predicting plant nutrient imbalances associated with variable soil fertility and it is usually based on analysis of index plant parts such as the youngest fully expanded leaf (YFEL). Recent use of the YFEL to diagnose plant nutrient status of Leucaena leucocephala subsp. glabrata (leucaena) pastures has given unreliable results. Two field trials, one irrigated and one dryland, were conducted in subtropical Queensland to investigate the effect of index leaf selection, plant phenology and environmental factors (ambient temperature and water stress) on leaf nutrient concentrations. The YFEL was identified as the best plant part to sample because it was readily identifiable and had consistent concentrations of most nutrients compared to older and younger leaves provided specific conditions were met when sampling. At both sites there was significant (P  0.75% DM were likely to be >21 days in age and should not be used for the diagnosis of plant nutrient status. It was concluded that leaf analysis could be used to confidently assess leucaena plant nutrient status provided the YFEL were sampled from actively growing plants in vegetative development that had received rainfall/irrigation in the preceding 28 days and were <21 days of age.