Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR) is a major pest of maize that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR, but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using carbon-11 with positron emission tomography, root autoradiography and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth which are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of glutamine. Finally, the regrowth zones of WCR attacked roots show an increase in glutamine turnover which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin flux network as a promising marker for induced WCR tolerance.

Nitrogen transfer within and between plants through common mycorrhizal networks (CMNs)

Mycorthizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH4+ and NO3-, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope N-15 is discriminated against during biogeochemical and biochemical processes. Differences in N-15 (atom%) or delta(15)N (parts per thousand) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both N-15 labeling and N-15 natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25similar to45 mum) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N-2-fixing mycorrhizal plants transferred to non-N-2-fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N-2-fixing mycorrhizal plants to N-2-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.

Reciprocal N ((NH4+)-N-15 or (NO3-)-N-15) transfer between nonN(2)-fixing Eucalyptus maculata and N-2-fixing Casuarina cunninghamiana linked by the ectomycorrhizal fungus Pisolithus sp.

Two-way N transfers mediated by Pisolithus sp. were examined by excluding root contact and supplying (NH4+)-N-15 or (NO3-)-N-15 to 6-month-old Eucalyptus maculata or Casuarina cunninghamiana grown in two-chambered-pots separated by 37 m screens. Mycorrhizal colonization was 35% in Eucalyptus and 66% in Casuarina (c. 29% N-2-fixation). Using an environmental scanning electron microscope, living hyphae were observed to interconnect Eucalyptus and Casuarina. Biomass and N accumulation was greatest in nodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, less in nonnodulated mycorrhizal Casuarina/mycorrhizal Eucalyptus pairs, and least in nonnodulated nonmycorrhizal Casuarina/nonmycorrhizal Eucalyptus pairs. In nonnodulated mycorrhizal pairs, N transfers to Eucalyptus or to Casuarina were similar (2.4-4.1 mg per plant in either direction) and were 2.6-4.0 times greater than in nonnodulated nonmycorrhizal pairs. In nodulated mycorrhizal pairs, N transfers were greater to Eucalyptus (5-7 times) and to Casuarina (12-18 times) than in nonnodulated mycorrhizal pairs. Net transfer to Eucalyptus or to Casuarina was low in both nonnodulated nonmycorrhizal (< 0.7 mg per plant) and nonnodulated mycorrhizal pairs (< 1.1 mg per plant). In nodulated mycorrhizal pairs, net transfer to Casuarina was 26.0 mg per plant. The amount and direction of two-way mycorrhiza-mediated N transfer was increased by the presence of Pisolithus sp. and Frankia, resulting in a net N transfer from low-N-demanding Eucalyptus to high-N-demanding Casuarina.

Timing, magnitude, and location of initial soluble aluminum injuries to mungbean roots

Despite a century's knowledge that soluble aluminum (Al) is associated with acid soils and poor plant growth, it is still uncertain how Al exerts its deleterious effects. Hypotheses include reactions of Al with components of the cell wall, plasmalemma, or cytoplasm of cells close to the root tip, thereby reducing cell expansion and root growth. Digital microscopy was used to determine the initial injuries of soluble Al to mungbean (Vigna radiata L.) roots. Roots of young seedlings were marked with activated carbon particles and grown in 1 mm CaCl2 solution at pH 6 for ca. 100 min (control period), and AlCl3 solution was added to ensure a final concentration of 50 muM Al (pH 4). Further studies were conducted on the effects of pH 4 with and without 50 muM Al. Four distinct, but possibly related, initial detrimental effects of soluble Al were noted. First, there was a 56-75% reduction in the root elongation rate, first evident 18-52 min after the addition of Al, root elongation continuing at a decreased rate for ca. 20 It. Decreasing solution pH from 6 to 4 increased the root elongation rate 4-fold after 5 min, which decreased to close to the original rate after 130 min. The addition of Al during the period of rapid growth at pH 4 reduced the root elongation rate by 71% 14 min after the addition of Al. The activated carbon marks on the roots showed that, during the control period, the zone of maximum root growth occurred at 2,200-5,100 mum from the root tip (i.e. the cell elongation zone). It was there that Al first exerted its detrimental effect and low pH increased root elongation. Second, soluble Al prevented the progress of cells from the transition to the elongation phase, resulting in a considerable reduction of root growth over the longer term. The third type of soluble Al injury occurred after exposure for ca. 4 h to 50 mum Al when a kink developed at 2,370 mum from the root tip. Fourth, ruptures of the root epidermal and cortical cells at 1,900-2,300 mum from the tip occurred greater than or equal to4.3 h after exposure to soluble Al. The timing and location of Al injuries support the contention that Al initially reduces cell elongation, thus decreasing root growth and causing damage to epidermal and cortical cells.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Lack of mycorrhizal autoregulation and phytohormonal changes in the supernodulating soybean mutant nts1007

Autoregulatory mechanisms have been reported in the rhizobial and the mycorrhizal symbiosis. Autoregulation means that already existing nodules or an existing root colonization by an arbuscular mycorrhizal fungus systemically suppress subsequent nodule formation/root colonization in other parts of the root system. Mutants of some legumes lost their ability to autoregulate the nodule number and thus display a supernodulating phenotype. On studying the effect of pre-inoculation of one side of a split-root system with an arbuscular mycorrhizal fungus on subsequent mycorrhization in the second side of the split-root system of a wild-type soybean (Glycine max L.) cv. Bragg and its supernodulating mutant nts1007, we observed a clear suppressional effect in the wild-type, whereas further root colonization in the split-root system of the mutant nts1007 was not suppressed. These data strongly indicate that the mechanisms involved in supernodulation also affect mycorrhization and support the hypothesis that the autoregulation in the rhizobial and the mycorrhizal symbiosis is controlled in a similar manner. The accumulation patterns of the plant hormones IAA, ABA and Jasmonic acid (JA) in non-inoculated control plants and split-root systems of inoculated plants with one mycorrhizal side of the split-root system and one non-mycorrhizal side, indicate an involvement of IAA in the autoregulation of mycorrhization. Mycorrhizal colonization of soybeans also resulted in a strong induction of ABA and JA levels, but on the basis of our data the role of these two phytohormones in mycorrhizal autoregulation is questionable.

Determining the age of adult wild dogs (Canis lupus dingo, C-l. domesticus and their hybrids). I. Pulp cavity: tooth width ratios

In order to determine the age of adult wild dogs, we compared two methods ( that of Thomson and Rose (TR method) and that of Knowlton and Whittemore (KW method)) of measuring and calculating pulp cavity : tooth width ratios on upper and lower canine teeth from 68 mixed-sex, known-age wild dogs of 9 months to 13 years of age reared at two localities. Although significant relationships ( P = 0.0001) were found between age and pulp cavity ratios by both methods, the TR ratio calculation and measurement showed heteroscedasity in error variance whereas the KW ratios had a more stable error variance and were normally distributed. The KW method also found significant differences between pulp cavity ratios between teeth of the upper and lower jaws ( P < 0.0001) and sex ( P = 0.01) but not geographic origin ( P = 0.1). Regressions and formulae for fitted curves are presented separately for male and female wild dogs. Males show greater variability in pulp cavity decrements with age than do females, suggesting a physiological difference between the sexes. We conclude that the KW method of using pulp cavity as a proportion of tooth width, measured 15 mm from the root tip and averaged over both upper canines, is the more accurate method of estimating the age of adult wild dogs.

Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere

Enhanced biodegradation of organic xenobiotic compounds in the rhizosphere is frequently recorded although the specific mechanisms are poorly understood. We have shown that the mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) is enhanced in soil collected from the rhizosphere of Trifolium pratense[e.g. maximum mineralization rate = 7.9 days(-1) and time at maximum rate (t(1)) = 16.7 days for 12-day-old T. pratense soil in comparison with 4.7 days(-1) and 25.4 days, respectively, for non-planted controls). The purpose of this study was to gain a better understanding of the plant-microbe interactions involved in rhizosphere-enhanced biodegradation by narrowing down the identity of the T. pratense rhizodeposit responsible for stimulating the microbial mineralization of 2,4-D. Specifically, we investigated the distribution of the stimulatory component(s) among rhizodeposit fractions (exudates or root debris) and the influence of soil properties and plant species on its production. Production of the stimulatory rhizodeposit was dependent on soil pH (e.g. t(1) for roots grown at pH 6.5 was significantly lower than for those grown at pH 4.4) but independent of soil inorganic N concentration. Most strikingly, the stimulatory rhizodeposit was only produced by T. pratense grown in non-sterile soil and was present in both exudates and root debris. Comparison of the effect of root debris from plant species (three each) from the classes monocotyledon, dicotyledon (non-legume) and dicotyledon (legume) revealed that legumes had by far the greatest positive impact on 2,4-D mineralization kinetics. We discuss the significance of these findings with respect to legume-rhizobia interactions in the rhizosphere.

Effect of root zone temperature on oilseed rape (Brassica napus) response to boron

Oilseed rape (Brassica napus) is sensitive to low boron (B) supply, and its growth response to B may be influenced by soil temperature. To test the relationship between B and temperature, oilseed rape (cv. Hyola 42) seedlings were grown at 10 degrees C (low) root zone temperature (RZT) with B supply from deficient to adequate B levels until growth of low B plants just began to slow down. Half of the pots were then transferred to 20 degrees C (warm) RZT for 11 days before they were moved back to 10 degrees C RZT for the final 4 days. Both plant dry mass and B uptake increased after plants were exposed to warm RZT. However, plant B deficiency was exacerbated by warm RZT in low B plants because of increased relative growth rate and shoot-root ratio without a commensurate increase in B uptake rate. It is concluded that RZT above the critical threshold for chilling injury in oilseed rape can nevertheless affect the incidence of B deficiency by altering shoot-root ratio and hence the balance between shoot B demand and B uptake.

Functional significance of dauciform roots: exudation of carboxylates and acid phosphatase under phosphorus deficiency in Caustis blakei (Cyperaceae)

Caustis blakei produces an intriguing morphological adaptation by inducing dauciform roots in response to phosphorus (P) deficiency. We tested the hypothesis that these hairy, swollen lateral roots play a similar role to cluster roots in the exudation of organic chelators and ectoenzymes known to aid the chemical mobilization of sparingly available soil nutrients, such as P. Dauciform-root development and exudate composition (carboxylates and acid phosphatase activity) were analysed in C. blakei plants grown in nutrient solution under P-starved conditions. The distribution of dauciform roots in the field was determined in relation to soil profile depth and matrix. The percentage of dauciform roots of the entire root mass was greatest at the lowest P concentration ([P]) in solution, and was suppressed with increasing solution [P], while in the field dauciform roots were predominately located in the upper soil horizons, and decreased with increasing soil depth. Citrate was the major carboxylate released in an exudative burst from mature dauciform roots, which also produced elevated levels of acid phosphatase activity. Malonate was the dominant internal carboxylate present, with the highest concentration in young dauciform roots. The high concentration of carboxylates and phosphatases released from dauciform roots, combined with their prolific distribution in the organic surface layer of nutrient-impoverished soils, provides an ecophysiological advantage for enhancing nutrient acquisition.

Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1-1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root. No significant differences between har1-1 mutant and wild-type plants were detected with respect to root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.

The effect of copper toxicity on the growth and morphology of Rhodes grass (Chloris gayana) in solution culture

A solution culture experiment was conducted to examine the effect of Cu toxicity on Rhodes grass (Chloris gayana), a pasture species used in mine site rehabilitation. The experiment used dilute, solution culture to achieve external nutrient concentrations which were representative of the soil solution, and ion exchange resins to maintain stable concentrations of Cu in solution. Copper toxicity was damaged plant roots, with symptoms ranging from disruption of the root cuticle and reduced root hair proliferation, to severe deformation of root structure. A reduction in root growth was observed at an external Cu concentration of

Effect of physical, chemical and light treatments on germination and growth of tissue-cultured coconuts

The acclimatization and ex vitro establishment of tissue cultured coconut plantlets regenerated either from zygotic or somatic embryos could result to serious losses. Although high germination rates can be achieved in vitro, the survival of zygotic embryo derived plantlets in soil is very low (0-30%). Hence, treatments that could promote development of good quality seedlings having well-developed shoot and root is needed to increase seedling survival ex vitro. The effect of physical, chemical and light quality treatments on germination and growth of coconut embryos and tissue-cultured seedlings respectively, was investigated. The germination of coconut embryos was promoted when placed in a liquid Euwens (Y3) medium and incubated using a roller drum. Gibberellic acid (GA3) significantly affected growth of seedlings as it promoted shoot elongation, shoot and root expansion, and fresh and dry weight increase. However, GA3 did not significantly affect germination. In addition, the blue, red and yellow light significantly affected growth of seedlings as it promoted leaf and shoot elongation, fresh and dry weight increase, and root and leaf production. These conditions could be used to improve the growth and survival ex vitro of tissue cultured coconuts.

Facilitating soil-and plant-water research through the use of TDR

The development of TDR for measurement of soil water content and electrical conductivity has resulted in a large shift in measurement methods for a breadth of soil and hydrological characterization efforts. TDR has also opened new possibilities for soil and plant research. Five examples show how TDR has enhanced our ability to conduct our soil- and plant-water research. (i) Oxygen is necessary for healthy root growth and plant development but quantitative evaluation of the factors controlling oxygen supply in soil depends on knowledge of the soil water content by TDR. With water content information we have modeled successfully some impact of tillage methods on oxygen supply to roots and their growth response. (ii) For field assessment of soil mechanical properties influencing crop growth, water content capability was added to two portable soil strength measuring devices; (a) A TDT (Time Domain Transmittivity)-equipped soil cone penetrometer was used to evaluate seasonal soil strengthwater content relationships. In conventional tillage systems the relationships are dynamic and achieve the more stable no-tillage relationships only relatively late in each growing season; (b) A small TDR transmission line was added to a modified sheargraph that allowed shear strength and water content to be measured simultaneously on the same sample. In addition, the conventional graphing procedure for data acquisition was converted to datalogging using strain gauges. Data acquisition rate was improved by more than a factor of three with improved data quality. (iii) How do drought tolerant plants maintain leaf water content? Non-destructive measurement of TDR water content using a flat serpentine triple wire transmission line replaces more lengthy procedures of measuring relative water content. Two challenges remain: drought-stressed leaves alter salt content, changing electrical conductivity, and drought induced changes in leaf morphology affect TDR measurements. (iv) Remote radar signals are reflected from within the first 2 cm of soil. Appropriate calibration of radar imaging for soil water content can be achieved by a parallel pair of blades separated by 8 cm, reaching 1.7 cm into soil and forming a 20 cm TDR transmission line. The correlation between apparent relative permittivity from TDR and synthetic aperture radar (SAR) backscatter coefficient was 0.57 from an airborne flyover. These five examples highlight the diversity in the application of TDR in soil and plant research.

Fossil trees in ancient fluvial channel deposits: evidence of seasonal and longer-term climatic variability

It has been established that large numbers of certain trees can survive in the beds of rivers of northeastern Australia where a strongly seasonal distribution of precipitation causes extreme variations in flow on both a yearly and longer-term basis. In these rivers, minimal flow occurs throughout much of any year and for periods of up to several years, allowing the trees to become established and to adapt their form in order to facilitate their survival in environments that experience periodic inundation by fast-flowing, debris-laden water. Such trees (notably paperbark trees of the angiosperm genus Melaleuca) adopt a reclined to prostrate, downstream-trailing habit, have a multiple-stemmed form, modified crown with weeping foliage, development of thick, spongy bark, anchoring of roots into firm to lithified substrates beneath the channel floor, root regeneration, and develop in flow-parallel, linear groves. Individuals from within flow-parallel, linear groves are preserved in situ within the alluvial deposit of the river following burial and death. Four examples of in situ tree fossils within alluvial channel deposits in the Permian of eastern Australia demonstrate that specialised riverbed plant communities also existed at times in the geological past. These examples, from the Lower Permian Carmila Beds, Upper Permian Moranbah Coal Measures and Baralaba Coal Measures of central Queensland and the Upper Permian Newcastle Coal Measures of central New South Wales, show several of the characteristics of trees described from modern rivers in northeastern Australia, including preservation in closely-spaced groups. These properties, together with independent sedimentological evidence, suggest that the Permian trees were adapted to an environment affected by highly variable runoff, albeit in a more temperate climatic situation than the modem Australian examples. It is proposed that occurrences of fossil trees preserved in situ within alluvial channel deposits may be diagnostic of environments controlled by seasonal and longer-term variability in fluvial runoff, and hence may have value in interpreting aspects of palaeoclimate from ancient alluvial successions. (C) 2001 Elsevier Science B.V. All rights reserved.