Leaching of genetically modified Pseudomonas fluorescens through intact soil microcosms: Influence of soil type

Summary The leaching of a genetically modified Pseudomonas fluorescens through soil was investigated using intact (undisturbed) soil microcosms over a 2-month period. The microcosms comprised large cylindrical cores of three contrasting soil types (a loamy sand, a sandy loam, and a clay loam) supporting a grass/clover sward. Late log-phase cells of Pseudomonas fluorescens containing lux genes encoding for bioluminescence were applied to the surface of the soil cores. Eighteen hours after application of the inocula, the microcosms were subjected to simulated rain events (9 mm per event) at 3-day intervals and leachates were analysed for the concentration of genetically modified bacteria. The lux-modified pseudomonads were detected immediately in leachate from the clay looam with a steady decline in the concentration of cells with time. Leaching of pseudomonads from the sandy loam and loamy sand only occurred over a few rain events and total recoveries from the leachate were lower than from the clay loam. Leaching patterns are discussed in relation to differences in structure of topsoil and subsoil, which determine the pathways of water flow, and to the matric potential at inoculation, which determines the pore-size classes into which cells were first introduced.     

Novel method for the study of the population dynamics of a genetically modified microorganism in the gut of the earthworm Lumbricus terrestris

Summary A laboratory microcosm study was used to investigate the survival and population dynamics of genetically modified microorganisms (GMM) in the gut of Lumbricus terrestris. Three methods of axenic earthworm production were investigated. An antibiotic mixture of streptomycin and cycloheximide was introduced either passively, mixed with sterile soil or cellulose, or actively, by teflon catheter. Worms treated by all methods lost weight but this was least for the catheter method which was also the only method to produce axenic earthworms. Axenic earthworms were used to determine the effect of competition with indigenous gut bacteria on ingested GMM. The GMM used was Pseudomonas fluorescens, strain 10586/FAC510, with chromosomally inserted Lux genes for bioluminescence, and chromosomal resistance to rifampicin. The bacteria were grown up to the mid-exponential phase before inoculation into earthworms. Bacteria in faecal material were enumerated by dilution plate counting using selective agar. The GMM were re-isolated from the casts of both antibiotic-treated and untreated earthworms. Lower concentrations of GMM and higher concentrations of indigenous bacteria in the casts of untreated compared to antibiotic-treated earthworms suggested that competition is a fundamental control on population dynamics of the introduced bacterial inocula ingested by earthworms.The catheter method, developed in this study, is proposed as a technique to contribute to the risk assessment of environmental release of GMM.     

Survival of bacteria introduced into soil by means of transport by Lumbricus rubellus

Four strains of bacteria, Rhizobium leguminosarum biovar trifolii, Pseudomonas fluorescens, Pseudomonas cepacia, and Flavobacterium sp., were introduced into loamy sand and then transported by earthworms of the species Lumbricus rubellus to uninoculated soil. Cell densities recovered from the earthworm gut and casts (both expressed per gram dry material) were significantly lower (up to 3 log units) than cell densities recovered from the inoculated soil. Total bacterial counts in casts were similar to those in the inoculated soil. In casts excreted into a sterile environment numbers of colony-forming units (CFU) increased, suggesting a favourable environment for growth. In casts excreted in a non-sterile environment, cell densities of introduced strains decreased. Casts therefore did not offer the introduced bacteria a protective micro-environment for survival in the bulk soil. Transport by worms of R. leguminosarum biovar trifolii and of P. fluorescens appeared to occur mostly by means of cast production; with the Flavobacterium sp. and P. cepacia a large proportion of the cells was possibly transported on the skin of earthworms.     

Effect of Hormogaster elisae (Oligochaeta; Hormogastridae) on the stability of soil aggregates

The production and stability of soil aggregates produced by laboratory cultures of the endogeic earthworm Hormogaster elisae was studied using three different techniques: the determination of the soil mean weight diameter (MWD), the aggregate tensile strength, and by the Blanchart method, which involves three different tests. The MWD index of soils was higher in microcosms containing H. elisae. Tensile strength was significantly higher in earthworm casts than in naturally formed aggregates. The Blanchart method also showed aggregates produced by earthworms to be more stable. The results of all three methods concur in that aggregates produced by H. elisae are larger and more stable than those produced in control microcosms without earthworms.     

Movement of the protozoan pathogen Cryptosporidium parvum through three contrasting soil types

The potential for transfer of the protozoan pathogen Cryptosporidium parvum through soil to land drains and, subsequently, water courses following the application of livestock waste to land was monitored in the laboratory using simulated rainfall and intact soil cores. Following irrigation over a 21-day period, Cryptosporidium parvum oocysts applied to the surface of soil cores (initial inoculum concentration 1×10⁸ oocysts core^–1) were detected, albeit in low numbers, in the leachates from clay loam and silty loam soils but not in that from a loamy sand soil. Variations in leaching patterns were recorded between replicate cores. At the end of the study soil cores were destructively sampled to establish the location of oocysts remaining within the soil. Distribution within cores was similar in all three soil types. The majority (72.8+-5.2%) of oocysts were found in the top 2 cm of soil, with numbers decreasing with increasing depth to 13.2±2.8%, 8.39±1.4%, and 5.36±1.4% at depths of 10, 20, and 30 cm, respectively.     

Earthworms of different functional groups affect the fate of the Bt-toxin Cry1Ab from transgenic maize in soil

The fate of the insecticidal Cry1Ab protein from crop residues (leaves and roots) of the transgenic maize variety MON810 was studied in the presence and absence of two earthworm species (Lumbricus terrestris, Aporrectodea caliginosa; separate incubations) in soil microcosms. The recombinant Cry1Ab protein was quantified using a highly sensitive ELISA. Control microcosms received corresponding non-transgenic plant material. All earthworms survived in the microcosms over a period of 5 weeks, irrespective of whether they received MON810 or non-transgenic plant material. Weight loss was observed for both earthworm species, independent of the plant material or transgenic modification. A strong decline of immunoreactive Cry1Ab in plant residues (mean initial concentration approx. 5000 ng g⁻¹) of MON810 was observed in all treatments, but in microcosms with earthworms this decline was significantly higher with less than 10% of the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab concentrations in casts were only 0.1% of those found in remaining plant material of the respective microcosms. No immunoreactive Cry1Ab proteins were found in earthworm tissues (threshold of detection: 0.58 ng g⁻¹ fresh weight). No further decline was found for Cry1Ab concentrations in casts of A. caliginosa during a subsequent period of 3 months of incubation in bulk soil (<0.1 ng g⁻¹) after removal of the earthworms from the microcosms, while in casts of L. terrestris the concentration decreased from 0.4 to below 0.1 ng g⁻¹. In conclusion, this study demonstrates that earthworms enhance the decline of immunoreactive Cry1Ab proteins from maize residues.     

Effect of passage through the intestinal tract of detritivore earthworms (Lumbricus spp.) on the number of selected Gram-negative and total bacteria

Laboratory experiments were carried out to investigate the fate of bacteria during and after passage through the intestinal tract of detritivore earthworms. Earthworms (Lumbricus spp.) were fed with cattle dung inoculated 7 days previously with one of five different Gram-negative bacteria. Bacterial concentrations were determined 2 days later in dung and soil, and in gut material from different parts of the earthworm intestinal tract. A high percentage (28–82%) of the total bacteria (epifluorescence direct counts) in the earthworm gut content was culturable. The concentration of total heterotrophic aerobic bacteria did not vary significantly among the five different bacterial additions and the non-inoculated control. In earthworm casts the number of total heterotrophs per gram dry matter (2.1×10⁹) was higher than in soil (1.7×10⁸), but lower than in the dung (1.5×10¹⁰). The test-bacteria, however, showed different survival patterns along the earthworm intestinal tract. The concentrations of Escherichia coli BJ 18 and Pseudomonas putida MM 1 and MM 11 in earthworm casts were lower than in the ingested dung, while concentrations of Enterobacter cloacae A 107 and Aeromonas hydrophila DMU 115 in dung and casts were similar. Ent. cloacae, and to aminor extent E. coli, were reduced in numbers by several orders of magnitude in the pharynx and/or crop. In the hind gut, however, the concentration of Ent. cloacae had increased to the same level as in the ingested dung, while the concentration of E. coli remained low. Our observations indicate that the bacterial flora of ingested food materials changes qualitatively and quantitatively during gut transit.     

Leaching of genetically modified Pseudomonas fluorescens through organic soils: Influence of temperature,soil pH,and roots

Summary The effects of soil temperature and bulk soil pH on the vertical translocation of a genetically modified Pseudomonas fluorescens inoculum were studied in reconstituted soil microcosms, in the presence and absence of growing Lolium perenne roots. The inoculated microcosms received one rainfall event per day (5 mm h^-1 for 6 h) for 5 days and the resulting leachate was quantitatively assayed for the presence of the modified pseudomonad. Soil temperature affected the total number of modified pseudomonads detected in the leachate over the 5 days, with significantly lower numbers detected at 25°C compared to 5°C. The bulk soil pH also affected leaching of the inoculum, with significantly greater numbers detected in the effluent at pH 7.5 than at pH 4.5. In the absence of L. perenne, greater numbers of the modified pseudomonads were detected in the pH 7.5 soil after 5 days of leaching compared to soil at pH 4.5. L. perenne roots decreased the number of cells of the inoculum that were leached and detected in the soil after 5 days of leaching. In the soil microcosms used for the pH study the distribution of the inoculum remaining with the soil was altered by L. perenne roots. At each pH value the proportion of cells detected within the soil below the surface 2 cm of the microcosms was greater in the presence of L. perenne roots. The results of this study indicate that soil temperature, bulk soil pH, and the presence of root systems are important factors in determining the extent of inoculum translocation, and should be considered in the design and interpretation of field experiments.     

Combined effect of bioaugmentation and bioturbation on atrazine degradation in soil

Earthworms, because they change soil physical and chemical properties, are efficient engineers that act on soil microbial community and activity. Thus they may drive pollutant biodegradation in soil such as atrazine mineralization. We hypothesized that earthworms modify the abundance of indigenous soil bacteria and the fate and activity of atrazine-degraders in the soil they engineer by bioturbation. Two bacterial strains were used as bioaugmentation agents: Pseudomonas sp. ADP and Chelatobacter heintzii, which have acquired the capacity to metabolize atrazine by carrying plasmidic atz A, B, C, D, E, F and atzA, B, C, trzD genes, respectively. We analyzed the interactions between earthworms (Lumbricus terrestris) and the indigenous and atrazine-degrading (indigenous and inoculated) bacterial communities by quantifying the 16S rRNA and the atzA gene sequence copies numbers, respectively, in different earthworm microsites. The kinetics of atrazine mineralization were measured to link the bacterial community changes with the degradation function. Digestion by earthworms significantly impacted the number of indigenous bacteria and atrazine mineralization in bioaugmented soils. Regarding the fate of the two atrazine-degraders tested, Pseudomonas sp. strain ADP survived better within the 10 days of experiment than C. heintzii in the bulk soil but the surviving fraction of C. heintzii was still metabolically active and able to mineralize atrazine. A positive “burrow-lining” effect on the atzA sequence copies number was observed in soil whether bioaugmented with C. heintzii or not (i.e. native indigenous atzA) thereby indicating that burrow-linings form a specific ‘hot spot’ for atrazine-degraders. The present study is the first to report the role of earthworms in selecting native catabolic key-genes in soil (indigenous atzA). This catabolic gene selection through earthworm soil bioturbation could be important in sustaining the degradation (detoxification) function of soil.     

Enhanced microbial degradation of pentachlorophenol from soil in the presence of earthworms: Evidence of functional bacteria using DNA-stable isotope probing

This study investigated the effect of two earthworm species (Amynthas robustus Perrier and Eisenia fetida Savigny) on the soil microbial degradation of pentachlorophenol (PCP). PCP-degrading microbes were identified using DNA-stable isotope probing (SIP). The results showed that adsorption and fixing to soil particles and organic fractions dominated the fate of PCP in soil without any amendments. The inoculation of both earthworm species significantly enhanced soil PCP disappearance and basal respiration. The DNA-SIP results revealed that Klebsiella, Cupriavidus, Aeromonas, and Burkholderia spp. were present at higher relative abundances in [¹³C]-labeled-PCP-amended soil microcosms than [¹²C]-PCP-amended soil in the presence of A. robustus, indicating that these bacterial species were responsible for PCP assimilation. Cupriavidus and Aeromonas sp. were also detected in the earthworm gut before inoculation, and their relative abundance was affected by earthworms. These results demonstrated that earthworms can introduce functional bacteria into soils and increase the population of PCP-degrading bacteria, thereby accelerating soil PCP degradation.     

Nitrogen fixation and CO2 exchange in soybeans (Glycine max L.) inoculated with mixed cultures of different microorganisms

N₂ fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of biological nitrogen in plants as determined by the ¹⁵N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g^-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N₂ fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.     

Effects of earthworms on nitrogen mineralization

The influence of earthworms (Lumbricus terrestris and Aporrectodea tuberculata) on the rate of net N mineralization was studied, both in soil columns with intact soil structure (partly influenced by past earthworm activity) and in columns with sieved soil. Soil columns were collected from a well drained silt loam soil, and before the experiment all earthworms present were removed. Next, either new earthworms (at the rate of five earthworms per 1200 cm³, which was only slightly higher than field numbers and biomass) were added or they were left out. At five points in time, the columns were analyzed for NH ₄ ⁺ , NO ₃ ^– , and microbial biomass in separate samples from the upper and lower layers of the columns. N mineralization was estimated from these measurements. The total C and N content and the microbial biomass in the upper 5 cm of the intact soil columns was higher than in the lower layer. In the homogenized columns, the C and N content and the microbial biomass were equally divided over both layers. In all columns, the concentration of NH ₄ ⁺ was small at the start of the experiment and decreased over time. No earthworm effects on extractable NH ₄ ⁺ were observed. However, when earthworms were present, the concentration of NO ₃ ^– increased in both intact and homogenized cores. The microbial biomass content did not change significantly with time in any of the treatments. In both intact and homogenized soil, N mineralization increased when earthworms were present. Without earthworms, both type of cores mineralized comparable amounts of N, which indicates that mainly direct and indirect biological effects are responsible for the increase in mineralization in the presence of earthworms. The results of this study indicate that earthworm activity can result in considerable amounts of N being mineralized, up to 90 kg N ha^–1 year^–1, at the density used in this experiment.     

Effects of the earthworm Pontoscolex corethrurus on banana plants infected or not with the plant-parasitic nematode Radopholus similis

Radopholus similis is a worldwide endoparasitic nematode that greatly hampers banana (Musa acuminata, Cavendish subgroup) productivity. Earthworms are known to closely interact with above-ground and under-ground soil biota and particularly with plants and microfaunal communities. This study was aimed at investigating, under greenhouse conditions, the effects of the earthworm Pontoscolex corethrurus on banana growth and nutrient uptake, and assessing the influences of this earthworm on the development of an inoculated population of R. similis. Six-week-old tissue culture banana plants were submitted to four treatments: with P. corethrurus, R. similis, P. corethrurus+R. similis, and a control with no earthworms or nematodes. At the end of the experiment, the P. corethrurus treatments showed significantly higher leaf surface areas, shoot dry root weights, and root fresh weights than those without earthworms. This root growth enhancement probably contributed to the evident but non-significant decrease in the density of nematodes in the roots, even though earthworms did not reduce the total number of nematodes per whole root system. Moreover, the presence of earthworms slightly alleviated the severity of root damage. N bioavailability in the soil, along with N, Ca, and Mg content of banana plants, were also significantly increased in the presence of earthworms. Our results demonstrated that banana plant growth and nutrition were positively influenced by earthworms. Cropping practices that boost the development of earthworm communities in soil should therefore be promoted to enhance sustainability and to naturally alleviate nematode impact.     

The impact of earthworms on the abundance of Collembola: improvement of food resources or of habitat?

I assessed the direct influence of earthworm excretions, and the impact of earthworms through their action on the soil structure (increased macroporosity), on the population dynamics of the collembolan species Heteromurus nitidus. The intestinal content of Collembola arising from cultures on different soil types was observed, and two experimental cultures of H. nitidus were run: (1) a culture performed on an inert substrate supplied either with earthworm casts or with soil as food resource, (2) an experiment using microcosms with cores of two humus forms (moder and calcic mull), in the presence or absence of earthworms. The observation of gut contents revealed that H. nitidus feeds on excrements, the composition of which (ratio organic matter/mineral matter) varies according to the humus form where it lived. Slightly aged (10–15 days) organo-mineral casts of earthworms appeared to be a better food than calcic mull aggregates or organic material from moder. Densities of H. nitidus cultured in cores of calcic mull were higher than in moder, except when cores of moder were inhabited by an anecic earthworm for 2 months. The humus form strongly influenced populations of H. nitidus, firstly because densities of predators were higher in moder than in calcic mull, and probably also because of soil macroporosity. It was concluded that earthworms would affect predation on H. nitidus by creating a network of interconnected macropores in which Collembola can move and find shelter.     

Influence of soil ingestion by earthworms on the availability of potassium in soil: An incubation experiment

Summary An incubation experiment was conducted to study the changes that occur in the K status of soil due to earthworm activity. Samples of Tokomaru silt loam soil were inoculated with the common pasture earthworm species Aporrectodea caliginosa and incubated for 21 days. Aliquots of moist soil were analyzed for exchangeable K by leaching with neutral molar ammonium acetate at 1:50 soil solution ratio. Extraction with boiling 1 M nitric acid at 1:100 soil solution ratio for 20 min was used to determine available non-exchangeable K. The results indicated that the exchangeable K content increased significantly due to earthworm activity but nitric acid-extractable K did not change significantly. It is inferred that earthworms increase the availability of K by shifting the equilibrium among the forms of K from relatively un-available forms to more available forms in the soil chosen for the study.     

Influence of earthworms and leaf litter on edaphic variables in sewage-sludge-treated soil microcosms

Summary A series of 48 greenhouse soil microcosms were established and treated with combinations of sewage sludge, Acer saccharum leaves, and the earthworms Eisenia fetida and Lumbricus terrestris. The microcosms were incubated at constant moisture for 110 days. Samples were then taken and analysed for sludge depth, organic-matter content, and waterstable soil aggregates. Weights of surface leaves and weights and numbers of surviving earthworms were determined for each microcosm. L. terrestris significantly reduced sludge depth and the surface organic-matter content of microcosm soil and significantly increased percentages of 4-mm diameter water-stable aggregates. Leaf litter also significantly reduced sludge depth and increased 4-mm water-stable aggregates. E. fetida inhibited surface feeding by L. terrestris, reduced its 110-day survival rate, and inhibited the production of 4-mm water-stable aggregates in L. terrestris treatments. Numbers of E. fetida increased in L. terrestris treatments. Sludge depth, organic-matter content and water-stable aggregates were not significantly different from controls in E. fetida treatments.     

Effects of biomass and size of Millsonia anomala (Oligochaeta: Acanthodrilidae) on particle aggregation in a tropical soil in the presence of Panicum maximum (Gramineae)

Summary The present study assessed the effect of the tropical geophagous earthworm, Millsonia anomala, on the aggregate-size distribution of a sieved (2 mm), tropical ferruginous soil in the presence and absence of the perennial tropical grass Panicum maximum. The effect of two size groupings and graded biomass densities of M. anomala on soil aggregation was analysed in time and with depth in the containers within which the plants were grown. In the absence of earthworms, aggregation was rapid although limited (13.5% of soil as aggregates >2.0 mm), and probably arose from a combination of microbial activity and physical processes (interparticle bonding due to clays and other colloids). The roots of the test species contributed little to aggregation. In contrast, the effect of earthworm activity on soil aggregation was rapid and important. The effects of both biomass and, to a lesser extent, size were significant. After 79 days, aggregation reached a maximum with 3 g per container of small earthworms (ca. 59% of soil as aggregates >2.0 mm) and a minimum with 1 g per container of large earthworms (ca. 35% of aggregates >2.0 mm). Aggregation was considered to occur through three different mechanisms: (1) A rapid aggregation due to the interactions of colloids; (2) an intermediate aggregation due to a combination of unquantified processes related to earthworm activity (mucus secretion, development of fungus hyphae); (3) egestion of soil as earthworm casts which are stable macroaggregates.     

Effects of earthworms on decomposition and metal availability in contaminated soil: Microcosm studies of populations with different exposure histories

Population-specific differences in the responses of earthworms to simultaneous exposure to Cu and Zn were studied in microcosm experiments. Two populations of Aporrectodea caliginosa tuberculata (Eisen) with different metal exposure histories were chosen for the studies. Microcosms were prepared containing either uncontaminated soil or soils with low or high combined Cu/Zn -concentrations (79/139 or 178/311 mg kg⁻¹ dry mass of soil, respectively). Earthworms from each population were introduced to the microcosm treatments with some microcosms serving as controls without earthworms. One series of microcosms was destructively sampled after 16 weeks incubation in a climate chamber. Survival, growth, reproduction and decomposition by earthworms in each treatment were measured. An additional microcosm series was sampled for soil and earthworm measurements at four weeks intervals to determine temporal changes in the availability of metals in the soils and their accumulation into earthworms. Cu and Zn were sequentially extracted from the soil samples of both microcosm series to estimate mobility and availability of the metals in the soil. Earthworms with long-term exposure history to metal-contaminated soil seemed to tolerate higher soil metal concentrations than earthworms without earlier exposure. Both earthworms and metals affected soil respiration (CO₂ production) and nitrogen mineralization. In addition, earthworms seemed to decrease the mobility and bioavailability of metals in the soil through their burrowing activity.     

Effect of food type and placement on earthworm (Aporrectodea tuberculata) burrowing and soil turnover

Subsurface-dwelling Aporrectodea tuberculata, a common earthworm in Upper-Midwest (USA) agricultural fields, may be a significant component of agroeco-systems with regard to soil mixing and preferential transport of water and chemicals. In this study we looked at effects of food residue placement and food type on A. tuberculata burrowing and soil turnover in two-dimensional Evans box microcosms. Four food residue placements mimicked patterns induced by primary tillage and two food types, readily available and natural food sources, with no food as a control. An average earthworm population of 100 earthworms m^-2 was calculated to generate 1058 km ha^-1 of new burrows and turnover 7.9 Mg ha^-1 of soil in 1 week of activity at 20°C. Burrowing was random until food sources were encountered, at which time burrowing appeared to center around the food source.     

Interactions of Glomus clarum with Acetobacter diazotrophicus in infection of sweet potato (Ipomoea batatas), sugarcane (Saccharum spp.), and sweet sorghum (Sorghum vulgare)

Summary Spores of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus clarum obtained from sweet potatoes grown in soil inoculated with this fungus and with an enrichment culture of Acetobacter diazotrophicus contained A. diazotrophicus and several other bacteria, including a diazotrophic Klebsiella sp. Inoculation of micropropagated sweet potatoes with G. clarum and A. diazotrophicus enhanced spore formation in soil compared to VAM inoculation alone. Plants inoculated with VAM spores containing the bacteria showed additional increases in the number of spores formed within roots. A. diazotrophicus infected aerial plant parts only when inoculated together with VAM or when present within VAM spores. Micropropagated sugarcane seedlings inoculated with the same VAM spores containing the diazotrophs also contained much higher numbers of A. diazotrophicus in aerial parts than seedlings inoculated in vitro with the bacteria alone. When grown in non-sterile soil, the sugarcane seedlings again showed the greatest infection of aerial parts after inoculation with VAM spores containing the diazotrophs. This treatment also increased VAM colonization and the numbers of spores formed within roots. Similar effects were observed in sweet sorghum except that the aerial plant parts were not infected by A. diazotrophicus.