Delayed inoculation and competition of Rhizobium meliloti in annual Medicago species

Seed inoculation is frequently essential for annual Medicago establishment in Mediterranean dryland farming systems. As post-planting soil inoculation is often practiced when seed inoculation fails, the effect of delayed inoculation was investigated. Roots of Medicago rigidula (L.). All selection 716, and Medicago rotata Boiss. selection 2132, were initially pre-exposed to Rhizobium meliloti strains. Subsequently, roots were exposed to a secondary inoculum after 6, 48 and 168 h to simulate delayed inoculation. and subsequent establishment of others strains in the nodules were investigated. Combinations of highly effective and host compatible ICARDA strains (M15 and M53) and effective-ineffective strains (M15 and M28) were used to evaluate proportional nodulation responses. Plants were harvested after 6 weeks of growth. Nodules were assessed for distribution in the root system and for occupancy based on their differential resistance to kanamycin and streptomycin, and, in the case of ICARDA M28, on nodule characteristics. The strain ICARDA M15 was a better competitor than ICARDA M53 when applied in equal density in M. rotata, at zero time. While forming nodules with M. rigidula, ICARDA M53 was equally competitive under the same conditions. With ICARDA M15 as the primary inoculum, and ICARDA M53 delayed for 6, 48 and 168 h, the incidence of ICARDA M15 nodules increased on M. rigidula from 43% (at zero time) to 86, 94, and 97% and, on M. rotata, from 78% (at zero time) to 88%, 95%, and 98% for the three time delays, respectively. Pre-exposure of 2-day old M. rigidula seedlings to the ineffective strain ICARDA M28 as the primary inoculant was followed by significant nodule number increases (P 0.01) as compared with ICARDA M15. Nonetheless, when ICARDA M15 was the primary inoculum, M28 was able to produce about 35% of the nodules when applied at the 6 and 48 h time delays. However, with ICARDA M28 as the primary inoculum, followed by ICARDA M15, nodule occupancy of ICARDA M28 in M. rigidula increased from 40 (at zero time) to 75%, 80%, and 95% for the three time delays, respectively. The percentage of total nodulation by M28, applied at the three delay times, was markedly lower (10%, 5% and 1%, respectively) when M. rotata was pre-exposed to ICARDA M15. This suggested a host preference for ICARDA M15, even if applied as a late inoculum. Results indicate that the early events in the nodulation process of annual medics coupled with host-specificity factors are perhaps the most critical for competition among R. meliloti strains for nodule formation.     

Use of soil solarization to control root rots in gerberas (Gerbera jamesonii)

Summary An investigation was conducted during the summer months of 1986–1987 and 1987–1988 in Western Australia to evaluate the effect of soil solarization on the control of root rot of gerbera an also on the microbial and nutrient status of the soil. Infested soil cores were sampled from a site where root-rot was a severe problem and were removed to a non-infested site where they were subjected to soil solarization or fumigation. Soil solarization resulted in reduced root rot (root disease index 28.6%) in comparison to the untreated control (52.0%) 8 months after planting. Plants in the fumigated plots had 15.8% less disease than those in solarized plots. Solarization increased the total numbers of bacteria and actinomycetes, and the proportion of bacteria and fungi antogonistic to Fusarium oxysporum, F. solani and Rhizoctonia solani. The proportion of actinomycetes antagonistic to these fungi, however, did not differ between solarized and control soil treatments. There was a significant reduction in disease in plants grown in infested fumigated soil to which a 10% concentration of solarized soil had been added, suggesting the development of microbial suppression in solarized soil. Phytophthora cryptogea was eradicated to 30 cm by solarization as well as by fumigation. Solarization eliminated R. solani but not F. oxysporum to a soil depth of 10 cm. Solarization increased the levels of NO _n3 ^– -N and NH₄ ⁺-N in soil, but did not affect the concentrations of PO₄ ^3–, K⁺, Fe²⁺, organic C and pH. Yield (as number of flowers per plant) was increased by soil solarization and by fumigation.Increased yields and decreased disease severity in the solarized plots could have been caused by (1) a reduction in the infectivity of the infested soils, (2) an increase in the suppressiveness of the soil, and (3) an increased available of plant nutrients.     

Presence and dispersal of infective Frankia in peat and meadow soils in Sweden

Summary Use of the N₂-fixing grey alder, Alnus incana (L.) Moench, as a short-rotation crop for energy production is currently being explored. To evaluate the need for inoculation of alders, the distribution of infective propagules of Frankia in the soil at potential sites for alder plantations was examined. Uninoculated grey alder seedlings were grown in three types of soil. Frequent nodulation was found in a meadow soil which had been free from actinorhizal plants for nearly 60 years, but the alder seedlings failed to nodulate in peat soil from two different bog sites. One of these bogs had been exploited for peat and the surface layer of the peat had been removed, so that the soil samples were taken from deep layers of the peat. At the other site, an area of cultivated peat, there were no infective propagules of Frankia in plots without alders; the infective Frankia was present in plots only where it had been introduced by inoculated alders. There was no detectable air-borne dispersal of Frankia. Instead, water movement might account for the dispersal of Frankia in peat. Although the apparent absence of Frankia in these peat soils necessitates inoculation of alder seedlings before planting out, this makes it possible to introduce and maintain Frankia strains with selected beneficial characteristics, since there is no competition from an indigenous Frankia flora.     

Effect of temperature and soil moisture on the survival and symbiotic effectiveness of Frankia spp.

Comparison of the effects of temperature on the growth in culture (increase in protein) of Frankia showed that three strains isolated from Casuarina were more tolerant of high temperature (45°C) than a strain from Alnus rubra. Optimal temperatures for growth of the Casuarina strains were in the range 25–30°C. Growth of the Alnus strain was good at 25°C but poor at 37°C. High temperatures (35–40°C) during storage for 7 months of these Frankia strains in sand, inoculated initially with liquid culture or with Frankia incorporated into alginate beads and permitted to dry, resulted in substantial loss of infectivity for the host plant species. Loss in infectivity was greater with an Alnus Frankia strain than strains from Casuarina cunninghamiana, C. equisetifolia and C. junghuniana. Three Frankia strains from C. equisetifolia were incorporated into a sand/perlite mixture with three different moisture regimes (field moisture capacity – wet: watered and maintained at field capacity; watered to field capacity but then allowed to dry – moderately wet; or watered to half field capacity and then permitted to dry – dry) and then stored for 12 weeks at 25°C and 35°C. Assessment by the most probable number (MPN) technique of the infectivity of the sand mixture for nodulation of C. equisetifolia showed significant interactions between Frankia strain, temperature and soil moisture content. The infectivity of Frankia strains ORS020607 and UGL020602q was not affected by incubation in wet sand at 25°C but fell by more than half after 12 weeks in moderate and dry conditions. Changes in infectivity were similar when incubation was at 35°C. By contrast, the infectivity of UGL020603q fell substantially under all moisture conditions and at both temperatures. The data show the importance of screening for tolerance of both temperature and moisture content when selecting strains for preparation of inoculum for use in hot climates. Received: 25 January 1996     

Abundance of Frankia from Gymnostoma spp. in the rhizosphere of Alphitonia neocaledonica, a non-nodulated Rhamnaceae endemicto New Caledonia

The capacity of different soils of New Caledonia to induce nodulation in Gymnostoma poissonianum was studied. The soils were sampled under five Gymnostoma species, Alphitonia neocaledonica (a non-nodulated endemic Rhamnaceae) and Pinus caribea (an introduced species) growing in various ecological conditions. Using G. poissonianum as trap-host, we observed a higher abundance of Frankia from Gymnostoma spp. in the rhizosphere of A. neocaledonica as compared with bare soils and P. caribea rhizosphere. The nodulating capacity of A. neocaledonica rhizosphere was almost similar to that of the five Gymnostoma species (symbiotic host) studied in the same stations. In comparison, bare soils or rhizosphere of P. caribea had poor nodulating capacities. We isolated fourteen Frankia strains from nodules of G. poissonianum after baiting with the rhizospheric soils of five Gymnostoma and A. neocaledonica. Using the PCR/RFLP method, we confirmed the similarity with those already described. Frankia was abundant in the rhizosphere of A. neocaledonica in all the sites studied. One explanation could be a positive tropism of Frankia towards species belonging to families having nodulated species, which is the case of A. neocaledonica endemic in New Caledonia. We can suppose that the non-nodulated plants belonging to these families can excrete some chemical substances able to attract Frankia and to induce its proliferation.     

Soil suppressiveness and functional diversity of the soil microflora in organic farming systems

Arable fields of 10 organic farms from different locations in The Netherlands were sampled in three subsequent years. The soil samples were analysed for disease suppressiveness against Rhizoctonia solani AG2.2IIIB in sugar beet, Streptomyces scabies in radish and Verticillium longisporum in oilseed rape. In addition, a variety of microbial, chemical and physical soil characteristics were assessed. All data were correlated by multiple regression and multivariate analyses with the objective to find correlations between soil suppressiveness and biotic or abiotic soil characteristics. Significant differences in soil suppressiveness were found between the fields for all three diseases. Multiple regression indicated a significant correlation between suppressiveness against Rhizoctonia and the number of antagonistic Lysobacter spp., as well as with % active fungi and bacterial diversity. Grass-clover stimulated Rhizoctonia suppression as well as the presence of antagonistic Lysobacter spp. (mainly L. antibioticus and L. gummosus) in clay soils. Streptomyces suppression correlated with the number of antagonistic Streptomyces spp., % of active fungi and bacterial population size. The presence of antagonistic Streptomyces spp. correlated with a high fungal/bacterial biomass ratio. Verticillium suppression was only measured in 2004 and 2005, due to the inconsistent suppressiveness along the years. Nevertheless, a significant correlation with pH, potential nitrogen mineralization and bacterial biomass was found. Bacterial and fungal PCR-denaturing gel electrophoresis fingerprinting of bacterial and fungal communities, in general, did not significantly correlate with disease suppression. Highly significant explanatory factors of the composition of the dominating bacterial and fungal populations were % lutum, pH, C/N quotient, biomass and growth rate of bacteria. Additionally, the % of organic matter and years of organic farming were explaining significantly the composition of the bacterial population.Thus, significant correlations between several soil characteristics and suppressiveness of different soil-borne pathogens were found. For two of the three pathogens, suppression correlated with biotic soil characteristics combined with the presence of specific bacterial antagonists. Probably the soil suppressiveness measured in the organic fields is a combined effect of general and specific disease suppression.     

Nitrogen-fixing potential of Acacia mangium and Acacia auriculiformis seedlings inoculated with Bradyrhizobium and Rhizobium spp.

Summary Two Australian Acacia species, A. mangium and A. auriculiformis were inoculated in vitro with eight strains of Bradyrhizobium spp. and two strains of Rhizobium spp. On the two plant species, only Bradyrhizobium spp. strains formed effective N₂-fixing nodules. A. mangium, which nodulates effectively with a restricted range of Bradyrhizobium spp. strains, is a specific host compared to A. auriculiformis. A. auriculiformis is assumed to be a promiscuous host because it nodulates effectively with a wide range of Bradyrhizobium spp. strains. Nodule efficiency as expressed by the ratio of N₂ fixed to nodule dry weight appeared to be higher in A. auriculiformis (0.44–0.81) than in A. mangium (0.23–0.55).     

Impact of nodule damage by Rivellia angulata on N2-fixation,growth, and yield of pigeonpea (Cajanus cajan L. Millsp.) grown in a Vertisol

Summary Damage caused by Rivellia angulata larvae to pigeonpea root nodules at the ICRISAT center in India was greater in the crop grown on Vertisols (up to 86%) compared to that on Alfisols (20%). Attempts to quantify the field effects of nodule damage on growth and yield of pigeonpea in a Vertisol, involving many heavy applications of soil insecticides (aldrin and hexachlorocyclohexane) failed because the insecticides did not control the pest and adversely affected the growth of the pigeonpea and the subsequent crop of sorghum (Sorgorum bicolor L. Moench). The impact of nodule damage on pigeonpea growth, yield and nutrient uptake was successfully studied in greenhouse-grown plants at three N levels. In this pot study, artificial inoculation with Rivellia sp. led to substantial nodule damage (70%). The results of this damage were a significant overall reduction in nodule dry weight (46%), acetylene reduction activity (31%), total leaf area (36%), chlorophyll content of leaves (39%) and shoot dry weight (23%) 68 days after sowing. At maturity, Rivellia sp. infestation caused significant reductions in top dry weight (22%), root and nodule dry weight (27%), seed dry weight (14%), and total N (29%) and P uptake (19%). The problems and prospects of manipulating nodule damage so as to reduce N losses in pigeonpea are discussed.Submitted as JA No. 756 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean(Phaseolus vulgaris)is limited by the occurrence of damping off(rhizoctoniosis),which is caused by the fungus Rhizoctonia solani.However,the co-inoculation of plant growth-promoting rhizobacteria(PGPR)involved in biological control along with diatomic nitrogen(N2)-fixing rhizobia can enhance N nutrition and increase production.In this context,finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels.The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus,Paenibacillus,Burkholderia,and Pseudomonas with Rhizobium tropici CIAT 899,an N2-fixing rhizobial strain,on the biocontrol of damping off and growth promotion in common bean plants.Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola.The first experiment evaluated the potential of the 14 rhizobacterial strains,which were inoculated alone or in combination with CIAT 899,for the control of R.solani.The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply:co-inoculation with CIAT 899 at low mineral N supply(5.25 mg N mL^-1),low mineral N supply(5.25 mg N mL^-1),and high mineral N supply(52.5 mg N mL^-1).The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean.Co-inoculation of the strains UFLA 02-281/03-18(Pseudomonas sp.),UFLA 02-286(Bacillus sp.),and UFLA 04-227(Burkholderia fungorum)together with CIAT 899 effectively controlled damping off.For the common bean,mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293.Nodule endophytes UFLA02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean,promoting synergy with rhizobial inoculation and protection against disease.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA