Placement Of Inoculum Research Articles

Development and Evaluation of Laboratory Bioassays to Study Powdery Mildew Pathogens of Phlox In Vitro.

The genus Phlox consists of approximately 65 species that include some of the most prevalent ornamental plants in the temperate zone. These popular ornamentals are extremely susceptible to powdery mildew (PM) caused by the biotrophic fungi Golovinomyces magnicellulatus and Podosphaera sp. In this study, we used Phlox paniculata and P. glaberrima to develop a set of laboratory tools to study these pathogens in vitro, including a detached leaf and a micropropagated plantlet bioassay. We assessed pathogen growth under different experimental conditions, which included the use of four different media variations (1/2 MS medium amended with benzimidazole and tetracycline), three ages of pathogen culture (14, 18, and 22 days), three phenological stages of the host tissue (1st, 3rd, and 5th node leaves), placement of inoculum on both leaf surfaces (abaxial and adaxial), and three different inoculation techniques (single spore transfer, colony tapping, colony brushing). Detached P. paniculata leaves were successfully maintained on benzimidazole-amended 1/2 MS medium for up to 3 weeks. For both pathogens, the use of 18-day-old cultures resulted in a higher number of larger, higher sporulating colonies compared with 1-4 and 22-day-old cultures. The adaxial side of 3rd node leaves supported statistically significant more fungal growth compared with the adaxial side of 1st and 5th node leaves. Both pathogens also successfully infected micropropagated plantlets of P. glaberrima. These newly developed tools should facilitate in vitro studies on PM of Phlox and possibly be applicable to other ornamental species attacked by the same fungi.

Tracing native and inoculated Rhizophagus irregularis in three potato cultivars (Charlotte, Nicola and Bintje) grown under field conditions

Crop inoculation with arbuscular mycorrhizal fungi (AMF) is a promising option to increase plant yield. However, in most cases, the inoculated strains could not be traced in the field and their contribution to root colonization separated from native AMF. Therefore, there is no clear indication that growth promotion is strictly related to the inoculated isolates. Here, Rhizophagus irregularis MUCL 41833 was inoculated on three potato cultivars (Bintje, Nicola, Charlotte) under field conditions in Belgium. Inoculum was encapsulated into alginate beads and mycorrhizal infective potential (MIP) estimated with a dose-response relationship under greenhouse conditions before field experiment. Mitochondrial Large SubUnit (mtLSU) of inoculated R. irregularis MUCL 41833 was characterized to design haplotype- (inoculated R. irregularis haplotype) and species-specific (native or inoculated R. irregularis) markers. The magnitude of detection of the markers, determined by Real-Time quantitative PCR, was linked to the stage of colonization of potato cv. Bintje grown under greenhouse conditions. Under field conditions, the inoculant R. irregularis MUCL 41833 was detected at a very low level (between 10−5 and 10−7ng/ng total DNA) in a marginal number of plants, in contrast to native R. irregularis strains that were detected at higher levels (between 10−4 and 10−6ng/ng total DNA) in all plants of the three cultivars. This suggested that the inoculated strain was almost absent in the plants due either to the environmental conditions, competition with indigenous AMF or inadequate placement of inoculum. This was corroborated by the absence of growth promotion and differences in root colonization between inoculated versus non-inoculated potato plants. This study validated the mtLSU markers to detect/trace and quantify AMF inoculants as native strains in plants grown under field conditions and further supported that potato cultivars in the same field conditions differed in root colonization.

L-System model for the growth of arbuscular mycorrhizal fungi, both within and outside of their host roots.

Development of arbuscular mycorrhizal fungal colonization of roots and the surrounding soil is the central process of mycorrhizal symbiosis, important for ecosystem functioning and commercial inoculum applications. To improve mechanistic understanding of this highly spatially and temporarily dynamic process, we developed a three-dimensional model taking into account growth of the roots and hyphae. It is for the first time that infection within the root system is simulated dynamically and in a spatially resolved way. Comparison between data measured in a calibration experiment and simulated results showed a good fit. Our simulations showed that the position of the fungal inoculum affects the sensitivity of hyphal growth parameters. Variation in speed of secondary infection and hyphal lifetime had a different effect on root infection and hyphal length, respectively, depending on whether the inoculum was concentrated or dispersed. For other parameters (branching rate, distance between entry points), the relative effect was the same independent of inoculum placement. The model also indicated that maximum root colonization levels well below 100%, often observed experimentally, may be a result of differential spread of roots and hyphae, besides intrinsic plant control, particularly upon localized placement of inoculum and slow secondary infection.

Effects of inoculum depth on the activity of Rhizoctonia solani and on bean root rot development in sterile soil

The effects of different inoculum depths on the activity of Rhizoctonia solani and the development of root rot on beans were studied under greenhouse conditions. Inoculum was grown on rice grains and buried at depths of 0–5 cm, 510 cm, 10–15 cm and 15–20 cm. Experiments were carried out in PVC cylinders with removable rings, where the sowing was performed directly in infested soil, and in pots, where seeds were sown in the top layer (3 cm deep). Sowing was done immediately after soil infestation, as well as at 15, 30, 60 and 90 days after the soil infestation. In the cylinders, high disease severities were observed at all evaluation dates; emergence and plant weight were reduced by R. solani independently of the depth in which the inoculum was originally buried. The pathogen activity was not consistently reduced after 90 days in absence of host tissue in both experiments, although effects of R. solani on emergence and plant growth decreased over time. Moreover, the pathogen could very hardly be recovered from soil samples collected above the layer originally infested in the cylinders when it was deeply buried. In the pots, disease incidence and severity were high, and emergence and weight of plants were consistently reduced at all evaluation dates when the inoculum was confined in the upper 10 cm of soil. Damaging effects of R. solani on emergence and plant growth were reduced with deeper placement of inoculum. Emergence and plant weight were not affected at any evaluation date when the inoculum was buried deeper than 15 cm, although infected plants were frequently observed. As an epidemiological consequence, the primary inoculum for the next crops would be increased. The results indicate that the recommendation of deep burial of R. solani inoculum is not always valid, especially not in tropical lands, where it is common to prepare the soil twice or three times a year, considering that soil movements can deposit inoculum close to the soil surface.

A Simple Quantitative Procedure for Inoculation of Safflower with Teliospores of the Rust Fungus, Puccinia carthami.

A procedure is needed to determine susceptibility of safflower seedlings to teliospore inoculations of foreign accessions of Puccinia jaceae, a candidate agent for biological control of Centaurea solstitialis (yellow starthistle). The indigenous safflower rust pathogen, P. carthami, served as both model and basis for a future comparative study of these two rust fungi on safflower. The procedure involves inoculation of 2-day-old safflower seedlings on the hypocotyl near the base of the cotyledons with a known number of teliospores suspended in a 1-μl drop of a 0.1% water agar and wetting agent carrier. Advantages of this procedure include (i) quantification, by spore type, of inoculum applied to each seedling, (ii) precise placement of inoculum on test plants, and (iii) conservation of inoculum. Results were verified in studies with two safflower cultivars inoculated with 0, 15, 60, or 240 teliospores of either of two isolates of P. carthami. Plant height and dry weight data collected 6 weeks after inoculation revealed significant interactions for cultivar × inoculum concentration and for isolate × inoculum concentration. Similar results were achieved when safflower seeds were rolled in dry teliospores of P. carthami but not when seedlings grew through teliospore-infested sand. The latter two approaches (dry teliospore and infested sand inoculation) require much more inoculum and are only semiquantitative. The microliter drop procedure has potential usefulness in host range and quantitative studies involving other pathogens, including bacteria, fungi, and nematodes.

Competition among Sclerotinia sclerotiorum genotypes within canola stems

Populations of Sclerotinia sclerotiorum are often composed of multiple genotypes. In examining 35 naturally infected canola plants, 29 supported reproduction (i.e., sclerotium formation) by a single S. sclerotiorum genotype, as defined by the mycelial compatibility test. Only six plants supported reproduction by two genotypes. To test the hypothesis that infrequent multiple genotype infections were due to differences in virulence or competitive ability among isolates, four greenhouse experiments were conducted in which four isolates, representing three genotypes, were used in pairwise coinoculations of canola. There were no differences among the isolates in four virulence parameters. Mean reduction in sclerotial mass produced by a coinoculated isolate was calculated by comparison with the mean sclerotial mass of that isolate in the absence of competition, and used as the measure of competition. In all experiments, at least half of the coinoculation treatments resulted in reduced fungal reproduction for one or both of the coinoculated isolates, providing evidence of competitive differences. Generally, the magnitude of reproduction reduction was the same for each isolate in the pair. However, the magnitude was nonreciprocal when the more competitive isolate was given an advantage of early temporal arrival or spatial placement of inoculum at the lower position on the stem. Competitive differences among S. sclerotiorum isolates affecting reproduction represent one possible mechanism to explain temporal shifts in genotype frequencies. Key words: canola, competition, population structure, Sclerotinia sclerotiorum, virulence.

Effect of Strawberry Density on Dispersal ofColletotrichum acutatumby Simulated Rain

Studies were done with a rain simulator to evaluate the influence of plant density, inoculum source location, rain intensity, and their interactions on splash dispersal. Greenhouse-grown strawberry plants were mounted in support structures buried in soil to create artificial two-row canopies with 2-, 3-, 4-, and 8-cm intrarow spacings. Infected fruit placed either within or between the rows provided a source for spore dispersal by simulated rain, which was generated at constant intensities of 15 or 30 mm h -1 . Spore deposition, evaluated in sheltered petri plates exposed every 5 min over a period of 46 min, declined with increasing plant density when integrated over time at locations near the source. Deposition also declined with density when integrated over time and space for rows, area between rows, or the whole plot (P < 0.05). Within-row placement of inoculum and lower rain intensity reduced deposition compared to between-row placement and high rain intensity. Deposition gradients on plates between rows were described well by a negative exponential model. Slope parameters of spore deposition gradients between rows were not affected by any of the treatments, but the y-intercept was lowered by increased density, within-row placement of inoculum, and less intense rain (P < 0.05). Results suggest plant density affects both removal of spores from fruit and subsequent transport and may influence dispersal between sources and targets not within the canopy.

Effect of placement of inoculum in soil on infectivity and disease protection ability of a sterile red fungus

The effects of variations in the placement of inoculum in soil on the infectivity and disease protection ability of a sterile red fungus (SRF) was investigated. The ability of the SRF to spread in soil and to infect wheat plants either from pre-infected wheat plants or from an introduced source of inoculum was also examined. Experiments were carried out by placing the SRF inoculum at different distances (0, 1, 3, 5, 7 or 9 cm) from the plant and at different depths (0, 2, 4, 6, 8 or 10cm) in soil. Nylon mesh bags used in this study allowed the passage of fungal hyphae but prevented the free movement of wheat roots in soil. The furthest distance for the placement of the SRF inoculum for successful root colonization was found to e 3 cm. This distance was used to detect the differences in the period required to infect and colonize the wheat roots by the SRF in comparison with the take-all fungus. It was observed that the SRF maintains the initial advantage of early arrival on the wheat roots for protecting the plant from the take-all fungus. The SRF was also found to colonize and protect plant roots more effectively when it was placed below the seed at sowing than at a 3 cm distance from the plant. Although the potential for the SRF to spread from plant to plant exists, it may still be necessary to introduce the SRF inoculum close to each wheat seed at sowing.

Effect of Placement and Sequence of Inoculation with Fusarium oxysporum on its Interaction with Meloidogyne arenaria on Subterranean Clover

AbstractThe effect of the placement of inoculum of Fusarium oxysporum at two soil depths, and the sequences of inoculations with Meloidogyne arenaria and Fusarium oxysporum on root growth and development of root disease in Trifolium subterraneum L. (subterranean clover) were investigated. The timing of infection and the proximity of root tips of the host root system to infection by M. arenaria and F. oxysporum appeared to be the major determining factors of root growth and of disease development in plants exposed to the pathogens. Immediate contact of roots with F. oxysporum (where the fungus was placed at seed level of 10 mm depth) appeared to result in more severe effects on roots in the presence of the nematode than later infection by the fungus placed at 30 mm depth. The production of galls by the nematode and early infection by F. oxysporum at 10 mm depth resulted in a severe inhibition of root growth, particularly of the lateral roots. But no such growth inhibition was evident when F. oxysporum and M. arenaria were introduced together at the lower depth of 30 mm. The lowest density of M. arenaria inoculum was sufficient to cause severe root rot if F. oxysporum was present at the host seed level. With the fungus at 30 mm depth, however, the expression of root rot appeared to be influenced by the inoculum level of the nematode. In sequential inoculation with F. oxysporum or M. arenaria, the organism added 2 weeks later had little or no effect on root development. The first organism (M. arenaria or F. oxysporum) to infect the germinated seedlings was the main cause of root growth inhibition. The organism that came into contact with the roots 2 weeks later had little or no effect on the roots. Concurrent infection by F. oxysporum and M. arenaria resulted in less M. arenaria gall production on the tap root system than those added with the nematode alone or in advance of the fungus.

INOCULUM EFFICIENCY AND PATHOZONE WIDTH FOR TWO HOST‐PARASITE SYSTEMS

SummaryExperiments involving direct placement of inoculum at various distances from an infection court are used to estimate the probability of infection and/or disease of seminal roots of wheat by Gaeumannomyces graminis (Sacc.) Arx &amp; Olivier var. tritici Walker and of germinating seeds of radish by Rhizoctonia solani Kühn. Two concepts underlie the relationships under investigation. These are the pathozone, which is characterized by the furthest extent of inoculum from the host from which infection is recorded (z), and infection efficiency, which is characterized by two parameters α and β. The asymptotic infection efficiency, β, is estimated by the proportion of propagules that cause infection when placed in contact with the host; a is a measure of the decline in infection efficiency of propagules of inoculum with increasing distance from the host. Three non‐linear models are used to describe the profile of infection efficiency over distance.The sensitivity of the method of placement of inoculum in detecting treatment effects is demonstrated first by showing differences in response amongst two isolates of G. graminis and two times of observation. Maximal values of z for G. graminis ranged from 4 to 8 mm, depending upon the type of inoculum of G. graminis and the growth medium used. Values of z for R. solani and germinating radish seeds varied from 12 to 16 mm for propagules comprising fragments of chopped potato soil inoculum sieved to 1 to 2 mm.The amounts and nutritional basis of inoculum influenced the probability of infection and the parameters α, β and z. Thus, for G. graminis, estimated values for α−1, β and z were greater for inoculum comprising colonized millet grains than for colonized sand particles. When seedlings of radish were exposed to clumps of propagules of R. solani, the parameters β and z increased, while β decreased with increasing clump density. Reduction in propagule size to 0.5 to 1.0 mm reduced the proportion of infected hosts. When clumps of seedlings of radish were challenged with clumps of inoculum of JR. solani, the probability of infection of clumps increased with clump density, but there was no evidence for an effect on the probability of infection of individual seedlings within a clump. The relationship between the estimates of pathozone width and infection efficiency and models for infection of subterranean organs is discussed.

THE DEVELOPMENT OF ENDOMYCORRHIZAL ROOT SYSTEMS V. THE DETAILED PATTERN OF DEVELOPMENT OF INFECTION AND THE CONTROL OF INFECTION LEVEL BY HOST IN YOUNG LEEK PLANTS

SummaryLeek plants (Allium porrum L.) were grown on partly sterilized soil, in tall pots so that roots grew downwards unimpeded, with inoculum of the vesicular‐arbuscular mycorrhizal fungus Glomus mosseae (Nicol. &amp; Gerd.) Gerdemann &amp; Trappe placed either under the seedling or dispersed uniformly throughout the soil. The age of each individual root, and the distribution of infection in single roots were both determined in each of a series of sequential harvests.The development of the root systems was unaffected by placement of inoculum. Formation of adventitious roots continued up to 42 d, when branching commenced. The rate of initiation of roots was approximately exponential but more accurately fitted a logistic function. The increase with time in total length of root for the root system (Lt) was approximately exponential but the length of the single roots (Ltr) increased linearly.The total length of infection in whole root systems (Lir) also increased exponentially with time, but the rate of extension for the sum of the individual lengths of infected root (infection ‘segments’) in single roots (Ltr) was linear, with an apparent delay of approx. 5 d before infection could be observed.The rate of increase in Lir was very similar for placed (0.53 cm d1) and dispersed (0.58 cm d1) inocula, even though the number of infection segments per root differed widely. This suggests that the host controls the rate of growth of the fungus. On this basis, and assuming each root encountered a new propagule every 4 d, it was possible to predict the lengths and numbers of infection segments in single roots.The percentage infection in whole root systems and single roots, plotted against time, showed a delay, then a sharp rise to a final constant value. This pattern of development can be explained for single roots by simple arithmetical rules. Using these simple rules, and assuming that the rate of production of adventitious roots fitted either a logistic or exponential equation, it was possible to model the development of infection up to 42 d for a whole root system either algebraically or by numerical simulation.

Effects of Vesicular‐Arbuscular Mycorrhizae on Growth and Phosphorous Content of Three Agronomic Crops

AbstractSeveral placements of vesicular‐arbuscular (VA) mycorrhizal inoculum in soil were compared in a greenhouse study of the potential use of VA fungi for increasing yields of agronomic crops on low P‐status soils. Placement of VA mycorrhizal inoculum and rock phosphate was considered an integral treatment. Inoculation with VA fungi increased corn yields about 50% over the increases due to indigenous mycorrhizal fungi. Placements of inoculum in a layer 5 cm below the seeds or mixed with a 5‐cm layer of soil below the seeds were effective; while two other placements, with the seeds or in a band with rock phosphate, were ineffective in increasing yields. None of the treatments that used rock phosphate were as effective as concentrated superphosphate in increasing yields, which indicated that lack of P was still limiting plant growth in the rock phosphate treatments. There were no effects of VA mycorrhizal inoculation of soil on growth of soybeans (Glycine max L. Merrill ‘Harosoy 63’) and only a minor effect on sudangrass (Sorghum bicolor var. sudanense) for the placements that were tested.

Analysis of methods for growth detection in the search for extraterrestrial life.

In the search for life on other planets, experiments designed to detect the growth of microorganisms may prove to be definitive when coupled with chemical characterization and metabolic experiments. If organisms are not abundant, growth provides the only means for obtaining a large mass of biological material suitable for chemical compositional analyses and metabolic assays. Several methods of monitoring growth are described. Of these, optical monitoring in a unique system free of soil particles is advanced as the most appropriate. Theoretical problems related to the formulation of culture media are discussed, and several possible solutions are proposed. The sampling system, the type of monitoring, the size and placement of inoculum, and the medium volume and composition are contingent upon one another and must be integrated without sacrifice to the biological demands.

Analysis of Methods for Growth Detection in the Search for Extraterrestrial Life

In the search for life on other planets, experiments designed to detect the growth of microorganisms may prove to be definitive when coupled with chemical characterization and metabolic experiments. If organisms are not abundant, growth provides the only means for obtaining a large mass of biological material suitable for chemical compositional analyses and metabolic assays. Several methods of monitoring growth are described. Of these, optical monitoring in a unique system free of soil particles is advanced as the most appropriate. Theoretical problems related to the formulation of culture media are discussed, and several possible solutions are proposed. The sampling system, the type of monitoring, the size and placement of inoculum, and the medium volume and composition are contingent upon one another and must be integrated without sacrifice to the biological demands.

Effect of Soil Moisture Regime on Nodulation of Inoculated Peanuts1

AbstractA two‐year investigation on the effect of soil moisture regime (as conditioned by irrigation timing) and the depth of placement of inoculum on peanut inoculation was carried out in the northern Negev area of Israel. The inoculation was performed at planting time by introducing the bacterial suspension into the moist soil. The repeated drying‐out of the upper soil layers during irrigation cycles did not inhibit the process of nodulation. The placement of the inoculum at a depth of 12 cm resulted in poorer nodulation than at a depth of 3–4 cm, despite the higher soil moisture at the greater depth. Inoculation maintained a more favorable supply of nitrogen to the plants than did a 85 kg/1000 m2 application of ammonium sulfate and resulted in higher yields and better quality of peanuts.

Rhizosphere relationships of subterranean clover. I. Interactions between strains of Rhizobium trifolii.

A study was made of a number of effective and ineffective strains of Rhizobium trifolii in association with subterranean clover (Trifolium subterraneum L.), variety Bacchus Marsh. Where these were inoculated singly into sterilized soils in pot experiments conducted in the greenhouse, typical effective, ineffective, or intermediate plant reactions were obtained. Where more than one strain was inoculated, competition between strains took place and was reflected in nodule pattern and plant reaction. Some strains consistently failed to produce appreciable nodules in the presence of other rhizobia, some showed modification to varying degrees, and some were unaffected. Such behaviour was found to be closely linked with the ability of the strain to proliferate in the rhizosphere of the host plant, and placement of inoculum was shown to influence plant reaction. The concept of "incursion" as a property of a rhizobial strain is suggested. An incursive strain is one able to migrate from the initial site of inoculation and establish an adequate population in the root zone of the susceptible host despite the presence of active elements of the indigenous microflora and microfauna, including antagonistic and predatory forms. The property of incursion is independent of considerations of efficiency of nitrogen fixation or virulence for the host plant. Strains of rhizobia which are poorly incursive may fail to infect the host upon which they have been inoculated if subject to competition from indigenous rhizobia already established in the soil. The necessity of assessing properties of virulence and incursion as well as efficiency of nitrogen fixation in selecting rhizobia for purposes of commercial inoculation is stressed, and the methods of making such tests are discussed.