Daniel Real

The interactions of Rhizobium leguminosarum biovar trifolii in nodulation of annual and perennial Trifolium spp. from diverse centres of origin

The release of effective inocula for new perennial clovers into cropping zones where subterranean clover is important might compromise N2 fixation by this valuable annual clover if symbiosis between the new inoculants and subterranean clover is not optimal. To assist our understanding of the interactions between clovers and their microsymbionts, rhizobial strains and clovers from South and equatorial Africa, North and South America, and the Euro–Mediterranean regions were tested. Glasshouse-based studies of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum bv. trifolii associated with 38 genotypes of annual and perennial Trifolium spp. from these world centres of diversity were undertaken. Less than 7.5% of the perennial clover symbioses were effective whereas 40% of associations were effective for many of the annual clover species of Euro–Mediterranean origin. There was substantial specificity within the African clovers for effective nodulation. Rhizobial strains from the South American perennial T. polymorphum or from the African clovers were unable to nodulate subterranean clover effectively. Also, 7 of the 17 strains from these regions were unable to form nodules with the less promiscuous Mediterranean annual clovers, T. glanduliferum and T. isthmocarpum. Fifty-three of about 400 cross-inoculation treatments examined, which included annual and perennial clovers, were incapable of forming nodules, while only 65 formed effective nodules. There are 2 barriers to effective nodulation: a ‘geographic’ barrier representing the broad centres of clover diversity, across which few host-strain combinations were effective; and, within each region, a significant ‘phenological’ barrier between annual and perennial species. Clovers and their rhizobia from within the Euro–Mediterranean region of diversity were more able to cross the phenological barrier than genotypes from the other regions. It appears that only the relatively promiscuous clovers, whether annual or perennial, have been commercialised to date. The data indicate that, for perennial clovers, it will be a substantial challenge to develop inocula that do not adversely affect N2 fixation by subterranean clover and other annual clovers available commercially, especially if the perennial clovers were originally from Africa or America. Some future strategies for development of inoculants for clovers are proposed.

Evaluation of Perennial Forage Legumes and Herbs in Six Mediterranean Environments

There is an absence of drought tolerant herbaceous perennial forage legume and herb options other than lucerne (Medicago sativa L.) for environments with Mediterranean-like climates common in extensive areas of Southern Australia, the Mediterranean basin, and Chile. Therefore, a collection of 174 forage perennial legume and herb entries from 103 species and 32 genera was evaluated for adaptation in a diverse range of Mediterranean climatic environments in Southern Australia. The seasonal rainfall distribution varied from moderately to highly winter dominant with long term average annual rainfall ranging from 318 to 655 mm. The entries were rated for productivity and persistence over 3 yr. The 12 entries identified as the most promising for winter, summer, or all-year round production included Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata; Cichorium intybus L.; Cullen australasicum (Schltdl.) J.W. Grimes; Dorycnium hirsutum (L.) Ser.; Kennedia prostrata R. Br.; Lotononis bainesii Baker, Lotus pedunculatus Cav.; L. corniculatus L.; L. cytisoides L.; Medicago sativa subsp. sativa L.; Medicago sativa subsp. caerulea (Less. ex Ledeb.) Schmalh., and M. sativa subsp. falcata (L.) Arcang. These entries maintained production and persisted for the period of the evaluation, with the exception of C. intybus and L. corniculatus that declined in persistence over time. The potential role of these species in extensive grazing systems in Mediterranean climatic zones, their attributes and limitations, and current progress in developing them as useful forage plants was discussed.

Prioritisation of novel pasture species for use in water-limited agriculture: a case study of Cullen in the Western Australian wheatbelt

In the face of a drying climate, identification of perennial pasture species suited to low-rainfall agricultural areas is needed. Effective prioritisation of putative pasture species may be possible through the use of desktop methods that are commonly employed to investigate the effect of climate change on native plant populations or the weed risk potential of plants. Species of Cullen Medik. native to Australia may be useful as perennial pasture legumes. It is not known, however, if species have adaptations for growth in low rainfall regions like the wheatbelt of Western Australia (WA). We tested the hypothesis that some Cullen species would be adapted to the climate and soils of the low-rainfall wheatbelt of WA using an analysis of soils and climate niche models of herbarium and germplasm records of 16 perennial, herbaceous and Australian Cullen species. We identified nine Cullen species that had some likely adaptation to the climate in the wheatbelt of WA, five of which (C. australasicum, C. cinereum, C. discolor, C. patens and C. tenax) had widespread adaptation to the climate and showed some adaptation to the soils of the wheatbelt of WA, making these priorities for further evaluation as perennial pasture species. The methodology is useful to identify species with likely best adaptation to low rainfall regions, generate expectations of where these may be most successful and to identify gaps in existing germplasm collections.

Waterlogging tolerance and recovery of 10 Lotus species

Medicago sativa L. is the most widely sown perennial forage legume in farming systems of southern Australia. However, M. sativa lacks adaptation to winter waterlogged conditions. This constraint has highlighted the need for new perennial forage legumes adapted to winter waterlogged conditions and to locations where the summer is too dry for the survival of Trifolium repens L. and T. fragiferum L. To explore new perennial legume options suitable for these circumstances, 10 species of Lotus were evaluated for waterlogging tolerance including: two accessions of L. corniculatus L., four accessions of L. tenuis Waldst. & Kit., two accessions of L. pedunculatus Cav., two accessions of L. australis Andrew, three accessions of L. creticus L., three accessions of L. glaucus Sol., one accession of L. cruentus Court., one accession of L. argyrodes R.P. Murray, one accession of L. campylocladus Webb & Berthel and one accession of L. latifolius Brand. These were compared with the M. sativa cultivar Sceptre. The ability to grow in waterlogged conditions, and to recover, was assessed in a pot experiment conducted over 19 weeks. The Lotus species most tolerant of waterlogging were L. corniculatus, L. tenuis and L. pedunculatus. Each of these species developed aerenchyma, adventitious roots, surface roots and split stems at the base to improve oxygen transport into the roots. Significant intra-species variation was also found within each of the three tolerant species, highlighting the opportunity for genetic improvement. By comparison, the remaining Lotus species were susceptible to waterlogging and showed poor recovery, whereas M. sativa Sceptre partially recovered after waterlogging.

Lotus tenuis tolerates combined salinity and waterlogging: maintaining O2 transport to roots and expression of an NHX1-like gene contribute to regulation of Na+ transport

Salinity and waterlogging interact to reduce growth for most crop and pasture species. The combination of these stresses often cause a large increase in the rate of Na(+) and Cl(-) transport to shoots; however, the mechanisms responsible for this are largely unknown. To identify mechanisms contributing to the adverse interaction between salinity and waterlogging, we compared two Lotus species with contrasting tolerances when grown under saline (200 mM NaCl) and O(2)-deficient (stagnant) treatments. Measurements of radial O(2) loss (ROL) under stagnant conditions indicated that more O(2) reaches root tips of Lotus tenuis, compared with Lotus corniculatus. Better internal aeration would contribute to maintaining Na(+) and Cl(-) transport processes in roots of L. tenuis exposed to stagnant-plus-NaCl treatments. L. tenuis root Na(+) concentrations after stagnant-plus-NaCl treatment (200 mM) were 17% higher than L. corniculatus, with 55% of the total plant Na(+) being accumulated in roots, compared with only 39% for L. corniculatus. L. tenuis accumulated more Na(+) in roots, presumably in vacuoles, thereby reducing transport to the shoot (25% lower than L. corniculatus). A candidate gene for vacuole Na(+) accumulation, an NHX1-like gene, was cloned from L. tenuis and identity established via sequencing and yeast complementation. Transcript levels of NHX1 in L. tenuis roots under stagnant-plus-NaCl treatment were the same as for aerated NaCl, whereas L. corniculatus roots had reduced transcript levels. Enhanced O(2) transport to roots enables regulation of Na(+) transport processes in L. tenuis roots, contributing to tolerance to combined salinity and waterlogging stresses.

Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species

Six species of Trifolium (T. polymorphum Poir., T. riograndense Burkart, T. argentinense Speg., T. medium L., T. pratense L. and T. repens L.) were analyzed using inter-simple sequence repeats (ISSR) markers. Six selected primers generated 186 polymerase chain reaction (PCR) products exploring 112 loci in 34 genotypes analyzed with molecular sizes ranging from 200 to 1300 bp. These primers were able to discriminate among and within species, with the PCR products being on average 41.6% species-specific and 59.9% polymorphic at the within species level. Nuclear DNA content was determined by flow cytometry and revealed variation among species. The 1Cx genome size values were calculated and were found to range from 0.46 pg (T. pratense) to 0.96 pg (T. polymorphum). Genome size values of South American species were higher than those of Eurasiatic origin. The analyses of the molecular data grouped the six species in agreement with their geographical origin and clearly differentiate T. polymorphum from T. argentinense. The Eurasiatic group showed the highest average of species-specific bands (45.3%) and the South American group exhibited the highest amount of total bands (59.7). The highest level of intra-species polymorphisms was detected in T. argentinense (92.9%), followed by T. medium (89.5%).

Drought resistance at the seedling stage in the promising fodder plant tedera (Bituminaria bituminosa var. albomarginata)

Abstract. The perennial legume Bituminaria bituminosa (L.) C.H. Stirt. var. albomarginata (tedera) has been identified as a promising fodder plant for the southern Australian wheatbelt, but little is known about its drought resistance as a seedling. This study was conducted to (i) examine physiological and morphological responses to water stress of seedlings of tedera, in comparison with lucerne (Medicago sativa L.), biserrula (Biserrula pelecinus L.) and Afghan melon (Citrullus lanatus Thunb.), and (ii) investigate drought adaptation mechanisms of tedera seedlings. Seedlings were grown in a reconstructed field soil profile in pots in a glasshouse. By 25 days after sowing (DAS), plants of all species in the drought-stressed (DS) treatment had experienced water stress, with an average leaf relative water content (RWC) of 66% in DS compared with 79% in well-watered (WW) plants. Tedera, biserrula and Afghan melon maintained a higher RWC than lucerne. At 25 DAS, reductions in shoot dry matter in the DS treatment differed between species: 52% for Afghan melon, 36% for biserrula, 27% for lucerne, and no significant reduction for tedera. Paraheliotropic leaf angles of biserrula, lucerne and tedera were all higher in the DS treatment than in the WW treatment at 25, 32 and 52 DAS. This study revealed significant differences in rooting depth and stomatal conductance between the three legume species when under water stress, with tedera being the most drought-resistant. Traits that may allow tedera to survive a dry period following opening rains include vigorous seedling growth, early taproot elongation, effective stomatal control and paraheliotropic leaf movements.

Establishment, survival, and herbage production of novel, summer-active perennial pasture legumes in the low-rainfall cropping zone of Western Australia as affected by plant density and cutting frequency

Abstract. Herbaceous perennial legumes that can provide forage in the summer–autumn dry period are urgently required in Mediterranean climates to complement annual pastures and the perennial legume lucerne (Medicago sativa). This study evaluated the establishment, survival, and herbage production of tedera (Bituminaria bituminosa var. albomarginata) and Cullen spp. native to Australia. Two experiments were replicated at Buntine (warmer site) and Newdegate (cooler site) in the low-rainfall cropping zone (<350 mm average annual rainfall) of Western Australia from June 2008 to September 2010. In the first experiment, established by transplanting seedlings, survival and herbage production of two accessions each of B. bituminosa and C. australasicum were studied under densities of 1, 2, 4, 8, and 16 plants/m2 with 0, 1, 2, or 3 cuts in summer–autumn in addition to a winter–spring cut. In the second experiment, established from seed, emergence and survival of several accessions of B. bituminosa, C. australasicum, and M. sativa were studied, along with C. pallidum and C. cinereum. In the first experiment, B. bituminosa survived better than C. australasicum (70–80% v. 18–45%), especially at Buntine, but there was little impact of density or cutting frequency on survival. Plant death was highest during summer. Shoot dry weight (DW) accumulation varied greatly with site, year, and plant density. When rainfall was close to average, shoot DW was greater at Newdegate (B. bituminosa ≤7.4 t/ha, C. australasicum ≤4.5 t/ha) than at Buntine (≤2.3 t/ha), and both species produced much of their shoot DW in summer–autumn (e.g. 6 t/ha for B. bituminosa and 3 t/ha for C. australasicum at Newdegate). An early-summer cut reduced the DW that could be harvested later in summer–autumn. In the second experiment, emergence of B. bituminosa was either similar to, or higher than, emergence of the other species, being 43% at Buntine and 44% at Newdegate. Survival of B. bituminosa, compared with M. sativa, was similar at Buntine (13%) and slightly lower at Newdegate (14%). Emergence and survival of Cullen spp. varied among species and accessions, with survival of the best performing accession of C. australasicum (SA4966) similar to that of B. bituminosa and M. sativa at both sites. We conclude that B. bituminosa shows promise as a perennial summer forage for low-rainfall zones, with a density of 8–16 plants/m2 and cutting frequency of 3 cuts/year (i.e. cut twice in summer–autumn), while C. australasicum and C. pallidum warrant further study.

Variation in salinity tolerance, early shoot mass and shoot ion concentrations within Lotus tenuis: towards a perennial pasture legume for saline land

Perennial legumes are needed for productive pastures in saline areas. We evaluated 40 lines of Lotus tenuis for tolerance to salinity at both germination and vegetative growth stages. Salt tolerance during the early vegetative stage was assessed in a sand-tank experiment with NaCl concentrations of 0–450 mm NaCl for 5 weeks. Most L. tenuis lines were more salt tolerant and had at least 50% lower shoot Na+ plus Cl– (% dry mass (DM)) compared with some other common pasture legumes, Medicago sativa, M. polymorpha and Trifolium subterraneum. Within L. tenuis significant variation in salt tolerance was found, with C50 values (concentrations of NaCl that decreased shoot dry matter to 50% of control) ranging from ~100 to 320 mm. Shoot concentrations of Cl–, Na+ and K+ did not always correlate with salt tolerance; some tolerant lines had low shoot Na+ and Cl– (and thus better nutritive value), while others tolerated high shoot Na+ and Cl–. We also found variation within L. tenuis for salt tolerance of seeds, with lines ranging from 0 to 70% germination after recovery from a prior exposure to 800 mm NaCl for 15 days. There was no relationship between salinity tolerance of scarified seeds and subsequent growth of seedlings; therefore, testing of seeds alone would not be an appropriate screening method for salt tolerance in L. tenuis. This study of 40 L. tenuis lines has shown significant genetic variation for salt tolerance within this species, and we have identified key lines with potential to be productive in saltland pasture systems.

Evaluation and breeding of tedera for Mediterranean climates in southern Australia

Abstract. Tedera (Bituminaria bituminosa C.H. Stirton var. albomarginata and var. crassiuscula) has been identified as one of the most productive and drought-tolerant species of herbaceous perennial legumes based on 6 years of field evaluation in Western Australia in areas with Mediterranean climate and annual rainfall ranging from 200 to 600 mm. Importantly, tedera demonstrated broad adaptation to diverse soils, and some accessions have shown moderate levels of tolerance to waterlogging and salinity. Tedera exhibits minimal leaf shedding during summer and autumn. Economic modelling strongly suggests that giving livestock access to green tedera in summer and autumn will dramatically increase farm profit by reducing supplementary feeding. The breeding program (2006–12) evaluated the available genetic diversity of tedera for its field performance in seven nurseries with 6498 spaced plants in total covering a wide variation in rainfall, soils and seasons. Best overall plants were selected using a multivariate selection index generated with best linear unbiased predictors (BLUPs) of dry matter cuts and leaf retention traits. The breeding program also evaluated tedera for grazing tolerance, grazing preference by livestock, waterlogging tolerance, seed production, cold tolerance, disease susceptibility and presence of secondary compounds. Tedera is a diploid, self-pollinated species. Therefore, 28 elite parents were hand-crossed in several combinations to combine outstanding attributes of parents; F1 hybrids were confirmed with the aid of highly polymorphic, simple sequence repeat markers. The F1s were progressed to F4s by single-seed descent breeding. Elite parent plants were selfed for two generations to be progressed in the breeding program without hybridisation. Over time, selections from the crossing and selfing program will deliver cultivars of three ideotypes: (i) drought-tolerant, (ii) cold- and drought-tolerant, (iii) waterlogging- and drought-tolerant.