Response of subterranean clover cultivars to root rot fungi

Twelve widely grown cultivars of subterranean clover (Trifolium subterraneum) were screened both under controlled environment conditions for their resistance to five fungi commonly associated with root rot and under field conditions for their resistance to natural root infections. All cultivars showed decreased seedling survival (particularly from Pythium irregulare and Rhizoctonia solani), tap and lateral root rot (particularly from Fusarium avenaceum, P. irregulare, and R. solani) and reduced plant size (particularly from R. solani and P. irregulare). Individual cultivars generally differed in their response to the five pathogens and for any one pathogen there was generally a range of cultivar susceptibilities. Cultivars with the best resistance to individual root pathogens were identified. The results for the five individual pathogens under controlled conditions only showed correlation with field data for some of the parameters compared.     

Fungicidal drenches for control of root rot in subterranean clover

Fungicide drenches of benomyl, metalaxyl, iprodione, propamocarb, or thiram were applied to intact soil cores taken from known root rot affected fields in Western Australia, to control subterranean clover root rot. Metalaxyl was the most effective in reducing seedling damping-off. The most effective fungicide for reducing the level of rotting of both tap and lateral root systems of survixing plants varied from season to season at one site and varied between different sites in any one season with each fungicide giving a significant reduction in root disease on at least one occasion. Results suggest that different individuals or complexes of root pathogens were operative between seasons in any one site, and between sites for any one season. In some instances it appears that different individual root pathogens or pathogen complexes were operative on tap roots compared to lateral roots.     

Virus diseases of subterranean clover

Subterranean clover (Trifolium subterraneum) is grown as a pasture legume in several temperate regions of the world where the soils are acidic and infertile, and the rainfall is winter dominant and less than 600 mm annually. It is particularly important in southern Australia where more than 16 million ha have been sown with this species as the pasture legume component. Nine viruses have been recorded infecting subterranean clover in the field. These are alfalfa mosaic, bean yellow mosaic (pea mosaic), beet western yellows, clover yellow vein, cucumber mosaic, pea enation mosaic, soybean dwarf (subterranean clover red leaf), subterranean clover mottle and white clover mosaic. In addition there is an important problem referred to as subterranean clover stunt that was assumed to be caused by a virus but whose aetiology is still unknown. The importance of these diseases is reviewed and details on their epidemiologies are outlined together with details on progress towards their control and some comments on matters worthy of attention in the future. Reference is also made to several exotic viruses known to infect subterranean clover experimentally that could possible cause problems if introduced into Australia.     

Impact of Rhizobium nodules on Sitona hispidulus, the clover root curculio

The impact of nodules of Rhizobium on Sitona hispidulus (F.) developmental success was investigated in both laboratory and field situations. In the laboratory, alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) were grown with two nutrient solutions, one of which fostered and one of which inhibited nodulation. Neonate larvae were introduced onto the roots and survivorship and development monitored. Survivorship was greatly enhanced by the presence of nodules. Preference tests performed with neonate larvae indicated a strong attraction to root nodules. Field studies which used nitrogen fertilization to delay onset of nodulation in established alfalfa demonstrated that numbers of larvae and emerging adults were significantly lower in the nitrogen treated plots than in the control plots.

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Résumé L'influence des nodosités rhizobiales sur le développement et la survie de Sitona hispidulus a été étudiée en intervenant sur la nodulation de la plante. Au laboratoire, la luzerne (Medicago sativa) et le trèfle rouge (Trifolium pratense) se sont développés dans des pièces climatisées sur un substrat de sable/vermiculite et sur deux types de solutions nutritives (l'une avec azote, l'autre sans azote). La présence d'azote inhibe la nodulation des racines inoculées avec du Rhizobium. Des larves néonates ont été placées sur des racines et examinées après plusieurs semaines de croissance. La présence de nodules a eu un effet hautement significatif sur la survie larvaire. De plus, la présence de nodosités a effecté le développement larvaire, en augmentant le taux de développement sur luzerne et en le réduisant sur trèfle. Une expérience destinée à examiner la rapidité d'agrégation des larves néonates sur des racines nodulées ou non, révèle une forte préférence pour les racines à nodosités.Des expériences dans la nature ont permis de retarder la nodulation de la luzerne en dormance pendant l'hiver par apports d'azote; l'échantillonnage des larves et des émergences d'adultes a indiqué une diminution significative des populations dans les parcelles ayant reçu de l'azote.

     

The effect of sulfur supply on the root characteristics of subterranean clover and annual ryegrass

Summary The root characteristics and sulfur absorption of Trikkala sub-clover and Wimmera ryegrass were compared in a pot experiment in which plants were grown at two levels of sulfur supply (0 and 64 mgS/pot) for a period of 40 days after emergence.Ryegrass roots had a greater weight and root weight ratio, but a lower sulfur content than sub-clover roots at both levels of sulfur supply. Ryegrass roots were longer, had a greater length per unit weight, longer root hairs, and hence a larger volume and surface area than sub-clover roots. However, when the sulfur content in the whole plant was related to the root parameters, sub-clover absorbed a far greater amount of sulfur, irrespective of whether the content was expressed on the basis of the weight, length, volume or surface area of the roots.Sulfur application had relatively little effect on the morphology of roots of either species. However, whilst sulfur application increased the weight of both shoots and roots, it decreased the root weight ratio for sub-clover but not for ryegrass. Increasing sulfur supply reduced the total length of sub-clover roots but tended to increase the length of ryegrass roots. For both species, the length per unit weight of roots decreased, and the average root diameter increased, with increasing sulfur supply. Sulfur application decreased the volume and surface area of sub-clover roots, but increased these parameters in ryegrass roots.     

Influence of liming,inoculum level and inoculum placement on root colonization of subterranean clover

Arbuscular mycorrhizal (AM) fungi differ in their response to soil pH. Thus, change in soil pH may influence the relative abundance of mycorrhizal fungi inside roots. Root colonization by two AM fungi was studied in relation to addition of lime (CaCO3), quantity of inoculum and inoculum placement. Addition of CaCO3 to an acid soil decreased the colonization of roots by Acaulospora laevis but increased colonization by Glomus invermaium when both fungi were present. In acid soil (pH 4.7), almost all roots were colonized by A. laevis, while G. invermaium was dominant when soil pH was increased to pH 7.3. This occurred regardless of whether the inoculum was banded or mixed throughout the soil. There was no effect of CaCO3 on the relative abundance of fungi inside roots at intermediate rates of CaCO3 application (pH 5.3-6.3) when both fungi were inoculated together. In this experiment, both fungi colonized roots at all levels of CaCO3 when inoculated alone, except for A. laevis at the highest level of CaCO3. We conclude that soil pH affects the competitive ability of these two AM fungi during mycorrhiza formation primarily by affecting hyphae growth in soil and thus the relative abundance of hyphae at the root surface and subsequently inside the root.     

Effects of phosphorus application and mycorrhizal inoculation on root characteristics of subterranean clover and ryegrass in relation to phosphorus uptake

The effects of phosphorus (P) application and mycorrhizal inoculation on the root characteristics of subterranean clover and ryegrass were examined. Phosphorus application increased total root length, root surface area and root volume of both plant species. In contrast, mycorrhizal infection only affected the root characteristics of subterranean clover. Ryegrass took up more P than non-mycorrhizal subterranean clover at all levels of application. However, mycorrhizal infection only increased P uptake by subterranean clover and there was no difference in P uptake between ryegrass and mycorrhizal subterranean clover at low levels of P application. When the P uptake was expressed on the basis of any of the root characteristics, subterranean clover were superior to ryegrass suggesting that the greater uptake of P by ryegrass is not due to a higher efficiency in absorption of P from soil solution, but rather to a large root system.     

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.     

Effects of nutrient level on thinning and non-thinning crowding in even-aged populations of subterranean clover

Populations of subterranean clover were used to examine the effect of lowering nutrient supply on crowding in even-aged monospecific populations. Two hypotheses were being tested. Under one, termed ‘altered-speed’, reducing the nutrient supply to populations merely slows down the crowding process. Under the other, called ‘altered-form’, reducing the nutrient supply intensifies the crowding process (Morris & Myerscough 1984). The populations were grown at eight densities of sowing and three levels of nutrient supply. Non-thinning yield-density curves and self-thinning lines were fitted to the data. For the thinning populations, reducing the nutrient supply led to altered-form crowding, with thinning lines of reduced slope and intercept for total and shoot weight being observed in populations grown at lower levels of nutrient supply. For the non-thinning yield-density curves, lowering the nutrient supply did alter crowding effects, but not to the extent necessary to give full altered-form crowding. Rather, crowding effects intermediate between altered-speed and altered-form were observed. No altered-speed crowding was observed in this experiment. Comparison was made between the altered-speed crowding observed in other data (Hozumi & Ueno 1954: White & Harper 1970) with the altered-form crowding observed here and elsewhere (Hozumi & Ueno 1954). In these data, altered-form crowding was consistently associated with an increase in the proportion of root in the plants' growth as nutrient level was reduced, while in altered-speed crowding there was a complete lack of any such nutrient effect on root-shoot proportion. This was taken as evidence that under altered-speed crowding, variations in the level of nutrient supply did not lead to major change in the way that plants interfered with each other. For altered-form crowding, major change in the way plants interfered with each other's growth did occur as nutrient level fell.     

Effects of Gypsophila saponins on bacterial growth kinetics and on selection of subterranean clover rhizosphere bacteria

Plant secondary metabolites, such as saponins, have a considerable impact in agriculture because of their allelopathic effects. They also affect the growth of soil microorganisms, especially fungi. We investigated the influence of saponins on rhizosphere bacteria in vitro and in soil conditions. The effects of gypsophila saponins on the growth kinetics of rhizosphere bacteria were studied by monitoring the absorbance of the cultures in microtiter plates. Gypsophila saponins (1%) increased the lag phase of bacterial growth. The impact of gypsophila saponins on subterranean clover rhizosphere was also investigated in a pot experiment. The addition of gypsophila saponins did not modify clover biomass but significantly increased (twofold with 1% saponins) the weight of adhering soil. The number of culturable heterotrophic bacteria of the clover rhizosphere was not affected by the addition of gypsophila saponins. Nevertheless, the phenotypical characterization of the dominant Gram-negative strains of the clover rhizosphere, using the Biolog system, showed qualitative and quantitative differences induced by 1% saponins. With the addition of saponins, the populations of Chryseomonas spp. and Acinetobacter spp., the two dominant culturable genera of control clover, were no longer detectable or were significantly decreased, while that of Aquaspirillum dispar increased and Aquaspirillum spp. became the major genus. Aquaspirillum dispar and Aquaspirillum spp. were also the dominant rhizosphere bacteria of Gypsophila paniculata, which greatly accumulates these saponins in its roots. These results suggest that saponins may control rhizosphere bacteria in soil through rhizodeposition mechanisms.     

Antioxidative activity of copper in root nodules of yellow lupin plants

Copper nutrition inhibited lipid peroxidation in root nodules of yellow lupin plants at the early growth stages by about 50 %. The antioxidative activity of copper in the process of lipid peroxidation could be associated with Cu taking part in oxidative reaction of nodule catechol-like siderophores and its effect on iron accumulation and reactivity. The obtained results, for the first time, suggest that the ability of copper to inhibit lipid peroxidation in nodules could be considered as a major function for Cu requirements by symbiotic N₂ fixation in grain legume nodules.     

In vitro regeneration of commercial cultivars of subterranean clover

Regeneration of subterranean clover (Trifolium subterraneum L.) was achieved by both shoot organogenesis and somatic embryogenesis. Shoots derived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was sliced longitudinally into two halves and cultured on shoot induction medium. After 30 days, adventitious shoots were formed from the hypocotyl region while the radicle showed no development. Shoots were then subcultured onto shoot multiplication medium and finally onto a root initiation medium. Histological studies revealed that shoots arose de novo and did not originate from pre-existing meristems. In the second regeneration protocol, shoot apical meristems from young seedlings were induced to form callus. Following four to six weeks culture in the dark, somatic embryos appeared spontaneously on the calli. A majority of embryos had a well-defined root pole, two cotyledonary lobes, and were capable of germination, albeit at a low frequency. Regenerated plants obtained from both protocols appeared phenotypically normal.     

The structure of infection threads,bacteria and bacteroids in pea and clover root nodules

Summary The bacteria and infection threads of the pea root nodule were examined by light and electron microscopy. The bacteria in the infection thread are enclosed in a microcapsule. This capsule disappears when the bacterium is released into the host cytoplasm. The membrane envelope which surrounds the bacteria in the host cell is shown to be derived from the plant cell membrane. The infection of the host cell is by means of a process akin to pinocytosis and the bacteria are confined to vacuoles in the host cytoplasm. As each membrane envelope contains only one bacterium, the envelopes must divide with the bacteria. The bacteria increase 40fold in volume from the infection thread to the stage of the mature bacteroid. The mature infected host cell contains few organelles. The mitochondria become confined to the periphery of the cell. Differences of membrane structure in gram negative bacteria found by other workers have been attributed to fixation artifacts.     

Effects of seed phosphorus concentration on the emergence and growth of subterranean clover (Trifolium subterraneum)

Subterranean clover seed (Trifolium subterraneum cv. Dalkeith) with phosphorus concentrations of 0.75% (high P seed) and 0.48% (low P seed) and of uniform size (2.0–2.4 mm diameter) was used to measure the effect of seed P concentrations on seedling emergence and growth.Seedling emergence numbers were 35% greater for the high P seed, and this effect was independent of external P supply. High P seed also emerged more quickly than low P seed.Leaf emergence was faster and shoot dry weight was greater for seedlings grown from high compared with low P seed, but only when external P supply was deficient for plant growth. Phosphorus concentrations in the shoots of two-week old seedlings were 32–51% higher for high P seed, although by four weeks plants grown from high and low P seed had similar concentrations of P in their shoots. We suggest that establishing pastures using high P seed would improve both early and late season pasture production.