Arbuscular Mycorrhizal Fungi and Nutrition Determine the Outcome of Competition Between Lolium multiflorum and Trifolium subterraneum

Arbuscular mycorrhizal fungi (AMF) may affect competitive plant interactions, which are considered a prevalent force in shaping plant communities. Aiming at understanding the role of AMF in the competition between two pasture species and its dependence on soil nutritional status, a pot experiment with mycorrhizal and non-mycorrhizal Lolium multiflorum and Trifolium subterraneum was conducted, with manipulation of species composition (five levels), and nitrogen (N)- and phosphorus (P)- fertilization (three levels). In the non-mycorrhizal state, interspecific competition did not play a major role. However, in the presence of AMF, Lolium was the strongest competitor, with this species being facilitated by Trifolium. While N-fertilization did not change the competitive balance, P-fertilization gave Lolium, a competitive advantage over Trifolium. The effect of AMF on the competitive outcome may be driven by differential C-P trade benefits, with Lolium modulating carbon investment in the mycorrhizal network and the arbuscule/vesicle ratio at the cost of Trifolium.

A chronology of subterranean clover burr detachment mechanics and implications for seed harvestability

Seed retention has not been evaluated for subterranean clover ( Trifolium subterraneum L.), because its geocarpic seed-bearing burrs are currently harvested by suction systems. Development of improved harvest methods requires knowledge of subterranean clover seed retention characteristics and their changes with plant development. This study evaluates burr attachment and peduncle tensile strength during burr maturity until plant senescence across the three subspecies: subterraneum (cv. Dalkeith), yanninicum (cv. Monti) and brachycalycinum (cv. Mawson). Peduncle tensile strength was greater than burr-to-peduncle attachment strength for each subspecies, with peak mean peduncle strength 30–130% greater than peak mean burr-to-peduncle attachment strength. Both strength measurements decreased significantly (greater than 50% for each subspecies) as the plant senesced, which was associated with reductions in burr moisture content, and burr and peduncle diameters. Microscopy indicated a ductile to brittle transition as peduncles senesced, reducing energy absorption and increasing the likelihood of failure at defects. These results are important for the commercial production of subterranean clover seed and suggest it may be possible to harvest seed before plant senescence with dig-invert machinery, similar to that used for peanut harvesting. However, this approach would require harvesting prior to maximum seed development and the implications for seed viability and yield need to be further evaluated.

Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics in a Mediterranean apricot orchard

The soils of Mediterranean semiarid environments are commonly characterized by low levels of organic matter and mineral elements, as well as severe weed infestations, which, taken together, cause an intensive use of auxiliary inputs (tillage, fertilizers, herbicides). Although cover crops are recognized to sustainably improve soil health, the impact of Trifolium subterraneum L. cover cropping needs specific attention. This research investigates for the first time the effects over 4 years of T. subterraneum and spontaneous flora cover crops, after either incorporating their dead mulches into the soil or leaving them on the soil surface, on soil organic matter (SOM), macroelements, mineral nitrogen, microelements, and weed seedbank dynamics as indicators of soil quality in an apricot orchard. Compared to a conventional management control, the T. subterraneum cover crop with the burying of dead mulch into the soil increased the amount of SOM (+ 15%), ammoniacal (+ 194%) and nitric (+ 308%) nitrogen, assimilable P2O5 (+ 5%), exchangeable K2O (+ 14%), exchangeable Na (+ 32%), exchangeable K (+ 16%), Fe (+ 15%), Mn (+ 28%), Zn (+ 36%), and Cu (+ 24%), while it decreased the weed seedbank size (‒ 54%) and enhanced weed biodiversity. These findings suggest that T. subterraneum cover cropping may be an environment-friendly tool to enhance soil quality and limit auxiliary input supply in Mediterranean orchards.

Variation in waterlogging response among ecotypes of Trifolium subterraneum ssp. yanninicum and their relationships with site of origin

Background and AimsIn the annual pasture legume Trifolium subterraneum, ssp. yanninicum exhibits higher waterlogging tolerance than ssp. brachycalycinum and ssp. subterraneum. This study investigates waterlogging tolerance within ssp. yanninicum ecotypes and explores correlations with seedling phenotypic traits and site of origin eco-geographic variables.MethodsTwenty eight diverse ssp. yanninicum ecotypes collected from the Mediterranean region and four cultivars were grown in a controlled environment glasshouse. After 14 days of growth seedling traits were measured. After 21 days of growth, free-drained (control) and waterlogged treatments were imposed for 28 days. Eco-geographic variables were generated from ‘WorldClim’ using collection site locations.ResultsUnder waterlogging, shoot relative growth rate (RGR) ranged from 87–108% and root RGR ranged from 80–116% of controls. Waterlogging reduced shoot dry weight (DW) in four of 32 genotypes, while root DW was reduced in 13 genotypes. Leaf size was maintained, or even increased, under waterlogging in 31 genotypes. However, petiole length was more affected by waterlogging and has value as a waterlogging tolerance indicator. Waterlogging tolerance was not significantly correlated with seedling DW, flowering time or precipitation at the site of origin, while shoot growth under waterlogging had a positive correlation with summer temperatures at origin.ConclusionsGenotypes of ssp. yanninicum tolerated transient waterlogging and greater tolerance was observed among ecotypes, rather than cultivars. An easy-to-measure indicator of tolerance was found in petiole length reduction. This study highlights untapped genotypic variability for breeders to improve the productivity and persistence of ssp. yanninicum under waterlogging.

Predictive phytotoxic value of water-soluble allelochemicals in plant extracts for choosing a cover crop or mulch for specific weed control

Cover crops and mulches have become an alternative for soil management in vineyards due to the agronomic, environmental, and economic advantages, especially the possibility of weed control. Implicitly to this objective lies the idea of assessing the potential herbicide effect of the allelochemicals released by different cover crop and mulch species. With this objective, the present work evaluated the phytotoxic effects of 12 aqueous extracts of selected species with potential use as a cover crop or mulch: a Bromus species mixture (B. hordeaceus L. and B. rubens L.), Festuca arundinacea Schreb., Hordeum murinum L., H. vulgare L., Vulpia ciliata Dumort., Medicago rugosa Desr., M. sativa L., Trifolium subterraneum L., T. incarnatum L., Phacelia tanacetifolia Benth., Sinapis alba L., and Pinus sylvestris L.,on the germination and early growth of three troublesome weeds (Conyza bonariensis (L.) Cronquist, Aster squamatus (Spreng.) Hieron, and Bassia scoparia (L.) A. J.). The different in vitro bioassays showed that aqueous extracts of some species significantly inhibited or reduced germination and root and shoot growth of the target weed species in a dose-response manner. Germination of A. squamatus and C. bonariensis was reduced by 100-80% by the different extracts applied at 50% concentration, and completely blocked at 100% concentration, except for M rugosa extract to which both species showed less sensitivity. Root elongation of A. squamatus was absolutely inhibited under every extract and concentration, whereas C. bonariensis root growth showed only some tolerance to the crude extracts of F. arundinaceaand P. sylvestris. Bassia scoparia was relatively tolerant to the aqueous plant extracts, except for T. subterraneum crude extract, which reduced total germination by 80%; otherwise, B. scoparia showed higher general sensitivity of shoot growth than the other two weed species. The chemical profiles of phenolic compounds of the aqueous extracts were obtained and identified by HPLC-DAD, the phenolic profiles of H. murinum, V. ciliata, and M. rugosa being reported in this work for the first time. Using stepwise regression, the influence of certain phenolic compounds from the aqueous extracts on the germination and early growth of weeds was predicted. Among other significant compounds, the flavonoid naringenin identified in T. subterraneumaqueous extract at 8.09 µg·mL-1 was predicted to underlie its specific phytotoxicity on B. scoparia germination. These results support the use of cover crops and mulches in weed management and can help to select the most suitable species to adopt according to the target weed species. Highlights The phytotoxic nature of the aqueous extracts of twelve conventional and novel cover crops and mulch species was demonstrated on three troublesome weed species in vineyards. Phenolic acids and flavonoids of the twelve aqueous extracts were identified and quantified by HPLC-DAD, and, by regression analysis, some allelochemicals were postulated as responsible for the phytotoxic effects. The water-soluble phenolic profiles of three potential cover crops, namely Hordeum murinum, Vulpia ciliata, and Medicago rugosa, are reported for the first time. In vitro germination and early root growth of Conyza bonariensis and Aster squamatus were almost entirely restricted by any of the twelve plants' aqueous extracts and presumably by the joint action of their particular allelopathic compounds. Bassia scoparia germination was relatively much less sensitive to the extracts, except for Trifolium subterraneum, for which the flavonoid naringenin was predicted to underlie its specific phytotoxicity.

Effect of fungal endophytes on plant growth and nutrient uptake in Trifolium subterraneum and Poa pratensis as affected by plant host specificity

The introduction of well-adapted species, such as Trifolium subterraneum (subclover) and Poa pratensis (Kentucky bluegrass), might enhance the forage yield and quality of dehesa pastures for feeding livestock. However, the climatic hardness and poor soils in these agrosystems may limit plant establishment and development. Since fungal endophytes have been found to alleviate the environmental stresses of their host, the aim of this study was to assess the effect of five isolates on forage yield, nutritive value, and plant mineral uptake after their inoculation in the two abovementioned plant species. Two experiments were established (under greenhouse and field conditions) using plants inoculated with two isolates in 2012/2013 (Epicoccum nigrum, Sporormiella intermedia) and three isolates in 2013/2014 (Mucor hiemalis, Fusarium equiseti, Byssochlamys spectabilis). Fusarium equiseti (E346) increased the herbage yield of T. subterraneum under greenhouse conditions, and B. spectabilis improved the forage quality of T. subterraneum by reducing fiber content and of P. pratensis by increasing crude protein. S. intermedia increased the mineral uptake of Ca, Cu, Mn, Pb, Tl, and Zn in subclover, and M. hiemalis increased the uptake of K and Sr in Kentucky bluegrass. These results evidence the potential of the studied fungal endophytes to enhance herbage yield and nutritional value of forage, although further studies should include all of the target forage species as certain host specificity in the effect was observed.

Effect of Fungal Endophytes on Plant Growth and Nutrient Uptake in Trifolium Subterraneum and Poa Pratensis as Affected by Plant Host Specificity

The introduction of well-adapted species, such as Trifolium subterraneum and Poa pratensis, might enhance the forage yield and quality of dehesas pastures for feeding livestock. However, the climatic hardness and poor soils in these agrosystems may limit plant establishment and development. Since fungal endophytes have been found to alleviate the environmental stresses of their host, the aim of this study was to assess the effect of five isolates on forage yield, nutritive value and plant mineral uptake after their inoculation in the two above-mentioned plant species. Two experiments were established (under greenhouse and field conditions) using plants inoculated with two isolates in 2012/2013 (Epicoccum nigrum, Sporormiella intermedia) and three isolates in 2013/2014 (Mucor hiemalis, Fusarium equiseti, Byssochlamys spectabilis). Thus, F. equiseti (E346) increased the herbage yield of T. subterraneum under greenhouse conditions, B. spectabilis was found to improve the forage quality of T. subterraneum by reducing fibre content and of P. pratensis by increasing crude protein. Also, S. intermedia increased the mineral uptake of Ca, Cu, Mn, Pb, Tl and Zn in subclover and M. hiemalis, the uptake of K and Sr in Kentucky bluegrass. These results evidenced the potential of the studied fungal endophytes to enhance herbage yield and the forage nutritional value, although further studies should include all of the intended forage species as certain host specificity in the effect was observed.

Effect of Fungal Endophytes on Plant Growth and Nutrient Uptake in Trifolium Subterraneum and Poa Pratensis as Affected by Plant Host Specificity

The introduction of well-adapted species, such as Trifolium subterraneum and Poa pratensis, might enhance the forage yield and quality of dehesas pastures for feeding livestock. However, the climatic hardness and poor soils in these agrosystems may limit plant establishment and development. Since fungal endophytes have been found to alleviate the environmental stresses of their host, the aim of this study was to assess the effect of five isolates on forage yield, nutritive value and plant mineral uptake after their inoculation in the two above-mentioned plant species. Two experiments were established (under greenhouse and field conditions) using plants inoculated with two isolates in 2012/2013 (Epicoccum nigrum, Sporormiella intermedia) and three isolates in 2013/2014 (Mucor hiemalis, Fusarium equiseti, Byssochlamys spectabilis). Thus, F. equiseti (E346) increased the herbage yield of T. subterraneum under greenhouse conditions, B. spectabilis was found to improve the forage quality of T. subterraneum by reducing fibre content and of P. pratensis by increasing crude protein. Also, S. intermedia increased the mineral uptake of Ca, Cu, Mn, Pb, Tl and Zn in subclover and M. hiemalis, the uptake of K and Sr in Kentucky bluegrass. These results evidenced the potential of the studied fungal endophytes to enhance herbage yield and the forage nutritional value, although further studies should include all of the intended forage species as certain host specificity in the effect was observed.

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.