A sensitive method for measuring cyanide and cyanogenic glucosides in sand culture and soil

Summary Sensitive methods for measuring cyanide and cyanogenic glucosides in soil and sand culture have been developed. A microdiffusion technique is described which depends on the enzymic conversion of linamarin and lotaustralin to HCN, its release following acidification and incubation, and its detection in NaOH. Conditions for hydrolysis and HCN recovery have been optimised. The cyanide content of a silt loam soil (under non-cyanogenic wheat) was 5.47 nmol cyanide g^–1 air-dried soil whilst that in an organic soil under the cyanogenic bracken, Pteridium aqgilinum, was 12.2 nmol g^–1. Exudation of cyanogenic glucosides by linseed, Linum usitatissimum, was measured in plant growth tubes containing sand and a nutrient medium. Sterile plants exuded an average of 6.88 nmol glucosides plant^–1 week^–1 whilst, in contaminated tubes, the level fell to 4.72 nmol. Analysis of plant roots on each sampling occasion showed that 6.88 nmol was, on average, equivalent to 16.15% of the total root content of cyanogenic glucosides. There was a low but positive correlation between fresh weight of plant roots and the level of exuded glucosides. There was no evidence that plant roots produced free HCN.     

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.     

Phosphate solubilization by <Emphasis Type="Italic">Penicillium</Emphasis> spp. closely associated with wheat roots

In this study we found that Penicillium spp. exhibiting P-solubilizing activity are common both on and in the roots of wheat plants grown in southern Australian agricultural soils. From 2,500 segments of washed and surface-disinfested root pieces, 608 and 223 fungi were isolated on a selective medium, respectively. All isolates were screened for P solubilization on solid medium containing hydroxyapatite (HA); 47 isolates (5.7%) solubilized HA and were identified as isolates of Penicillium or its teleomorphs. These isolates were evaluated for solubilization of Idaho rock phosphate (RP) in liquid culture. Penicillium bilaiae strain RS7B-SD1 was the most effective, mobilizing 101.7 mg P l^–1 after 7 days. Other effective isolates included Penicillium simplicissimum (58.8 mg P l^–1), five strains of Penicillium griseofulvum (56.1–47.6 mg P l^–1), Talaromyces flavus (48.6 mg P l^–1) and two unidentified Penicillium spp. (50.7 and 50 mg P l^–1). A newly isolated strain of Penicillium radicum (KC1-SD1) mobilized 43.3 mg P l^–1. RP solubilization, biomass production and solution pH for P. bilaiae RS7B-SD1, P. radicum FRR4718 or Penicillium sp. 1 KC6-W2 was determined over time. P. bilaiae RS7B-SD1 solubilized the greatest amount of RP (112.7 mg P l^–1) and had the highest RP-solubilizing activity per unit of biomass produced (up to 603.2 g P l^–1 mg biomass^–1 at 7 days growth). This study has identified new isolates of Penicillium fungi with high mineral phosphate solubilizing activity. These fungi are being investigated for the ability to increase crop production on strong P-retaining soils in Australia.     

Influence of arbuscular mycorrhizal fungi and salinity on growth,biomass, and mineral nutrition of<Emphasis Type="Italic"> Acacia auriculiformis</Emphasis>

The effect of salinity on the efficacy of two arbuscular mycorrhizal fungi, Glomus fasciculatum and G. macrocarpum, alone and in combination was investigated on growth, development and nutrition of Acacia auriculiformis. Plants were grown under different salinity levels imposed by 0.3, 0.5 and 1.0 S m^-1 solutions of 1 M NaCl. Both mycorrhizal fungi protected the host plant against the detrimental effect of salinity. The extent of AM response on growth as well as root colonization varied with fungal species, and with the level of salinity. Maximum root colonization and spore production was observed with combined inoculation, which resulted in greater plant growth at all salinity levels. AM fungal inoculated plants showed significantly higher root and shoot weights. Greater nutrient acquisition, changes in root morphology, and electrical conductivity of soil in response to AM colonization was observed, and may be possible mechanisms to protect plants from salt stress.     

Reduction of bacterial growth by a vesicular-arbuscular mycorrhizal fungus in the rhizosphere of cucumber (Cucumis sativus L.)

Summary Cucumber was grown in a partially sterilized sand-soil mixture with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum or left uninoculated. Fresh soil extract was places in polyvinyl chloride tubes without propagules of mycorrhizal fungi. Root tips and root segments with adhering soil, bulk soil, and soil from unplanted tubes were sampled after 4 weeks. Samples were labelled with [³H]-thymidine and bacteria in different size classes were measured after staining by acridine orange. The presence of VAM decreased the rate of bacterial DNA synthesis, decreased the bacterial biomass, and changed the spatial pattern of bacterial growth compared to non-mycorrhizal cucumbers. The [³H]-thymidine incorporation was significantly higher on root tips in the top of tubes, and on root segments and bulk soil in the center of tubes on non-mycorrhizal plants compared to mycorrhizal plants. At the bottom of the tubes, the [³H]-thymidine incorporation was significantly higher on root tips of mycorrhizal plants. Correspondingly, the bacterial biovolumes of rods with dimension 0.28–0.40×1.1–1.6 m, from the bulk soil in the center of tubes and from root segments in the center and top of tubes, and of cocci with a diameter of 0.55–0.78 m in the bulk soil in the center of tubes, were significantly reduced by VAM fungi. The extremely high bacterial biomass (1–7 mg C g^-1 dry weight soil) was significant reduced by mycorrhizal colonization on root segments and in bulk soil. The incorporation of [³H]-thymidine was around one order of magnitude lower compared to other rhizosphere measurements, probably because pseudomonads that did not incorporate [³H]-thymidine dominated the bacterial population. The VAM probably decreased the amount of plant root-derived organic matter available for bacterial growth, and increased bacterial spatial variability by competition. Thus VAM plants seem to be better adapted to compete with the saprophytic soil microflora for common nutrients, e.g., N and P, compared to non-mycorrhizal plants.     

Effect of manganese-reducing rhizosphere bacteria on the growth of Gaeumannomyces graminis var. tritici and on manganese uptake by wheat (Triticum aestivum L.)

Summary Five bacterial strains capable of Mn reduction were isolated from the rhizosphere of plants growing in different South Australian soils. They differed in their Mn-reducing capacity. The antagonism of these strains compared to the imported strain 2–79 (from the United States) against Gaeumannomyces graminis var. tritici was tested in agar and in a soil sandwich experiment at different Mn²⁺ concentrations in the soil. In addition, wheat seeds were coated with the different strains and with MnSO₄ or with MnSO₄ only in order to investigate their effect on plant growth and Mn uptake. With one exception, all strains inhibited the growth of G. graminis in agar, but to different degrees. In contrast, only two strains significantly inhibited the growth of the fungus in the soil. The hyphal density was decreased more than the hyphal length. The Mn²⁺ concentration in the soil also had a marked effect on fungal growth; low Mn concentrations slightly increased while high Mn concentrations strongly decreased the fungal growth. Seed treatment with MnSO₄ only (+Mn) increased Mn uptake above that of the control (no seed treatment). Only the weakest Mn reducer on agar significantly increased plant growth and Mn uptake from soil in comparison with the Mn treatment. One strain was tested as seed coating without adding MnSO₄; it increased the plant growth to an extent similar to the Mn treatment. Increasing the Mn uptake by plants may be one of the growth-promoting effects exerted by rhizosphere bacteria.     

Cadmium bioavailability to Nicotiana tabacum L., Nicotiana rustica L., and Zea mays L. grown in soil amended or not amended with cadmium nitrate

Summary Mature (flowering) tobacco (Nicotiana tabacum cv. PBD6, Nicotiana rustica cv. Brasilia) and maize (Zea mays cv. INRA 260) plants were grown in an acid sandy-clay soil, enriched to 5.4 mg Cd kg^–1 dry weight soil with cadmium nitrate. The plants were grown in containers in the open air. No visible symptoms of Cd toxicity developed on plant shoots over the 2-month growing period. Dry-matter yields showed that while the Nicotiana spp. were unaffected by the Cd application the yield of Z. mays decreased by 21%. Cd accumulation and distribution in leaves, stems and roots were examined. In the control treatment (0.44 mg Cd kg^–1 dry weight soil), plant Cd levels ranged from 0.4 to 6.8 mg kg^–1 dry weight depending on plant species and plant parts. Soil Cd enrichment invariably increased the Cd concentrations in plant parts, which varied from 10.1 to 164 mg kg^–1 dry weight. The maximum Cd concentrations occurred in the leaves of N. tabacum. In N. rustica 75% of the total Cd taken up by the plant was transported to the leaves, and 81% for N. tabacum irrespective of the Cd level in the soil. In contrast, the Cd concentrations in maize roots were almost five times higher than those in the leaves. More than 50% of the total Cd taken up by maize was retained in the roots at both soil Cd levels. The Cd level in N. tabacum leaves was 1.5 and 2 times higher at the low and high Cd soil level, respectively, than that in N. rustica leaves, but no significant difference was found in root Cd concentrations between the two Nicotiana spp.Cd bioavailability was calculated as the ratio of the Cd level in the control plants to that in the soil or as the ratio of the additional Cd taken up from cadmium nitrate to the amount of Cd applied. The results showed that the plant species used can be ranked in a decreasing order as follows: N. tabacum > N. rustica > Z. mays.     

Inter-relation between descriptors and morphine yield in Asian germplasm of opium poppy Papaver somniferum

A set of 208 Indian and two Thai germplasm accessions of opium poppy Papaver somniferum were assessed for variation in 17 morphological characters, seed yield and content and yield of morphine from capsules and peduncles. The germplasm was found to be highly variable for all the characters evaluated. In the harvested peduncles and capsules, 13% was peduncle straw, 61% seeds and the rest capsule husk. The peduncle and capsule straw yields ranged between 0.6–2.2 and 1.4–5.3 g plant^-1, respectively. Morphine content in the peduncle varied between about 0.001–0.24% and that in the capsule from 0.02 to 1.05%. On average basis morphine content in the capsule husk was more than 9-fold higher than the peduncle straw. The plant morphine yields from peduncles and capsules ranged between 1.2 and 28.6 mg plant^-1. Four accessions yielded more than 20 mg of morphine plant^-1. Among these, in one of the accessions about 13% of the morphine was contributed by the peduncle. The plants of high morphine yielding accessions were generally small in height, and bore white flowers and large sized ungrooved capsules with a small number of seeds, on a large peduncle.     

Mycorrhizae, biocides, and biocontrol 3. Effects of three different fungicides on developmental stages of three AM fungi

The effects of biocide use on nontarget organisms, such as arbuscular mycorrhizal (AM) fungi, are of interest to agriculture, since inhibition of beneficial organisms may counteract benefits derived from pest and disease control. Benomyl, pentachloronitrobenzene (PCNB) and captan were tested for their effects on the germination and early hyphal growth of the AM fungiGlomus etunicatum (Becker & Gerd.),Glomus mosseae (Nicol. & Gerd.). Gerd. and Trappe andGigaspora rosea (Nicol & Schenck) in a silty-clay loam soil placed in petri plates. Application of fungicides at 20 mg active ingredient (a.i) kg^–1 soil inhibited spore germination by all three AM-fungal isolates incubated on unsterilized soil for 2 weeks. However, fungicides applied at 10 mg a.i. kg^–1 soil had variable effects on AM-fungal isolates. Fungicide effects on germination and hyphal growth of G.etunicatum were modified by soil pasteurization and CO₂ concentration in petri plates and also by placing spores below the soil surface followed by fungicide drenches. Effects of fungicides on mycorrhiza formation and sporulation of AM fungi, and the resulting host-plant response, were evaluated in the same soil in associated pea (Pisum sativum L.) plants. Fungicides applied at 20 mg a.i. kg^–1 soil did not affect the root length colonized byG. etunicatum, but both benomyl and PCNB reduced sporulation by this fungus. Benomyl and PCNB reduced the root length colonized byG. rosea at 48 and 82 days after transplanting. PCNB also reducedG. mosseae-colonized root length at 48 and 82 days, but benomyl only affected root length colonized byG. mosseae at the earlier time point. Only PCNB reduced sporulation byG. mosseae, consistent with its effect on root length colonized by this fungus. captan reduced the root length colonized by G. rosea at 48 days, but not at 82 days, and reduced colonization byG. mosseae at 82 days, but not at 48 days. Captan did not affect sporulation by any of the fungi.G. rosea spore production was highly variable, but benomyl appeared to reduce sporulation by this fungus. Overall,G. etunicatum was the most tolerant to fungicides in association with pea plants in this soil, andG. rosea the most sensitive. Benomyl and PCNB were overall more toxic to these fungi than captan. Interactions of AM fungi and fungicides were highly variable and biological responses depended on fungus-fungicide combinations and on environmental conditions.     

15N studies on the transfer of legume-fixed nitrogen to associated cereals in intercropping systems

Summary An attempt has been made to estimate quantitatively the amount of N fixed by legume and transferred to the cereal in association in intercropping systems of wheat (Triticum aestivum L.) — gram (Cicer arietinum L.) and maize (Zea mays L.) —cowpea (Vigna unguiculate L.) by labelling soil and fertilizer nitrogen with ¹⁵N. The intercropped legumes have been found to fix significantly higher amounts of N as compared with legumes in sole cropping if the intercropped cereal-legume received the same dose of fertilizer N as the sole cereal crop. But when half of the dose of the fertilizer N applied to sole cereal crop was received by intercropped plants, the amount of N fixed by legumes in association with cereals was significantly less than that fixed by sole legumes. Under field conditions 28% of the total N uptake by maize (21.2 kg N ha^–1) was of atmospheric origin and was obtained by transfer of fixed N by cowpea grown in association with maize. Under greenhouse conditions gram and summer and monsoon season cowpea have been found to contribute 14%–20%, 16% and 32% of the total N uptake by associated wheat and summer and monsoon maize crops, respectively. Inoculation of cowpea seeds with Rhizobium increased both the amount of N fixed by cowpea and transferred to maize in intercropping system.     

Effectiveness of two arbuscular mycorrhizal fungi on concentrations of essential oil and artemisinin in three accessions of Artemisia annua L.

A field experiment was conducted to study and compare the effectiveness of two arbuscular mycorrhizal fungi (AMF), Glomus macrocarpum (GM) and Glomus fasciculatum (GF) on three accessions of Artemisia annua. The AM inoculation significantly increased the production of herbage, dry weight of shoot, nutrient status (P, Zn and Fe) of shoot, concentration of essential oil and artemisinin in leaves as compared to non-inoculated plants. The extent of growth, nutrient concentration and production of secondary plant metabolites varied with the fungus–plant accession combination. The mycorrhizal dependency of the three accessions was related to the shoot: root ratio. Comparing the two fungal inoculants in regard to increase in essential oil concentration in shoot, the effectiveness of GF was more than that of GM. While in two accessions, GM was more effective in enhancing artemisinin concentration than GF. Increase in concentration of essential oil was found to be positively correlated to P-status of the plant. Conversely, no correlation was found between shoot-P and artemisinin concentration.     

Role of Azospirillum brasilense nitrate reductase in nitrate assimilation by wheat plants

Summary The nitrogen metabolism of wheat plants inoculated with various Azospirillum brasilense strains and nitrate reductase negative (NR^–) mutants was studied in two monoxenic test tube experiments. The spontaneous mutants selected with chlorate under anaerobic conditions with nitrite as terminal electron acceptor fixed N₂ in the presence of 10 mM NO₃ ^– and were stable after the plant passage. One strain (Sp 245) isolated from surface-sterilized wheat roots produced significant increases in plant weight at both NO₃ levels (1 and 10 mM) which were not observed with the NR^– mutants or with the two other strains. Similar effects were observed in a pot experiment with soil on dry weight and total N incorporation but only at the higher N fertilizer level. In the monoxenic test tube experiments plants inoculated with the mutants showed lower nitrogenase activities than NR⁺ strains at the low NO₃ ^– level (1 = mM) but maintained the same level of activity with 10 mM NO₃ ^– where the activity of all NR⁺ strains was completely repressed. The nitrate reductase activity of roots increased with the inoculation of the homologous strains and with the mutants at both NO₃ ^– levels. At the low NO₃ ^– level this also resulted in increased activity in the shoots, but at the high NO₃ ^– level the two homologous strains produced significantly lower nitrate reductase activity in shoots while the mutants more than doubled it. The possible role of the bacterial nitrate reductase in NO₃ ^– assimilation by the wheat plant is discussed.     

Crop residue and Collembola interact to determine the growth of mycorrhizal pea plants

The effects of collembolan grazing on arbuscular mycorrhizal (AM) fungi and plant growth were studied in a controlled experiment utilizing a mix of AM fungi and the dominant collembolan species (Isotoma sp.) indigenous to the experimental soil. Collembolan (+/– Col) effects were examined in the presence and absence of crop residue (+/– Litter) incorporated into the experimental soil. Significant interactions between collembolans and crop residue occurred for mycorrhizal colonization of roots and plant growth. In the absence of crop residue, collembolans reduced root length colonized by AM fungi, total plant dry mass and seed pod yield. However, in the presence of crop residue, collembolans had no effect on root colonization by AM fungi, and increased total plant mass and pod yield. Crop residue increased root colonization by AM fungi, numbers of bacteria and saprophytic fungi (colony forming units), small- (<5 m) and large- (>5 m) diameter hyphal lengths in soil, and the final population of collembolans in soil. Collembolans reduced both small- and large-diameter hyphae in soil and the number of saprophytic fungi (colony forming units, p =0.052). Feeding preference experiments conducted in vitro showed that Isotoma sp. preferred to graze on mycorrhizal roots over nonmycorrhizal roots when given no other food choice. However, when crop residue was added as a food choice, Isotoma sp. showed a clear feeding preference for crop residue. We conclude that collembolan grazing on mycorrhizae can be detrimental to plant growth when other fungal food sources are limited, but grazing on mycorrhizal fungi does not occur when ample organic matter and associated saprophytic fungi are present in soils.     

Agricultural potential of Mediterranean grain and forage legumes: Key differences between and within Vicia species in terms of phenology, yield, and agronomy give insight into plant adaptation to semi-arid environments

In order to identify key adaptive traits which affect productivity in Mediterranean grain and forage legumes and simultaneously determine the agricultural potential of a wide range of Vicia species, germplasm collected from the wild throughout the eastern Mediterranean was grown under semi-arid conditions in Tel Hadya, Syria (313 mm growing season rainfall). These included species currently in use in Mediterranean agriculture, such as V. sativa L., as well as those more widely used in the past-such as V. ervilia L., but also a broad selection from Section Narbonensis (B. Fedtsch. ex Radzhi) Maxted, including V. narbonensis L, V. johannis Tamamsch., V. hyaeniscyamus Mout., V. serratifolia Jacq., and V. kalakhensis Khattab et al. V. faba, a near relative of the taxa in Section Narbonensis, was included as a domesticated control. Where applicable, a representative range of subspecies was used. Accessions were chosen from a wide range of habitats in terms of latitude (31.02–40.72 decimal^o), longitude (27.1–43.17 decimal^o), altitude (20–1510 m), rainfall (180–1700 mm/yr) and soil depth (5–50 cm) in order to maximise diversity within species. Agricultural potential was determined by measuring seed, hay and biological yield, as well as agronomic traits such as harvest index, standing crop height, and seed size. The comparative influence of phenology and key agronomic traits such as plant habit and seed size on productivity varied tremendously between species, depending on their reproductive strategies. In V. sativa and V. ervilia, the smaller seed species which rely on long vegetative phases and growing seasons to accumulate sufficient biomass to set seed, and in which there was comparatively little agronomic variation, phenology had a large impact on yield. In early emerging taxa such as V. ervilia and V. s. subsp. sativa, with built-in long vegetative phases and growing seasons, seed yield was negatively correlated with flower ing (r = –0.86 to –0.88), whereas the opposite was the case for later emerging taxa such as V. s. subsp. nigra (L.) Ehrh. (r = 0.95). Within V. narbonensis and relatives, the larger seeded Vicia species which rely on more conservative reproductive strategies where high seedling vigour associated with large seeds enables the species to enter reproductive phases relatively early, phenology had a much smaller impact on yield than did variation of key traits such as seed weight, plant habit and pod shattering. Among the undomesticated germplasm harvest indices ranged from 0.09–0.31, hay yields from 0.1–3.4 t/ha, seed yield from 0–2.0 t/ha, and dry matter at maturity from 1.6–6.5 t/ha. Sub-specific taxonomy was crucial in determining agronomic potential. V. narbonensis var. aegyptiaca Kornhuber ex Asch. et Schweinf. showed the most potential, combining an upright habit, large seeds (212 mg) and tendency to retain intact pods after maturity, with the highest yield, harvest index and crop height of all the wild Vicia species. V. sativa subsp. sativa, V. ervilia and V. narbonensis var. narbonensis were less productive, but still showed agricultural potential. The smaller seeded V. narbonensis, var. affinis, var. jordanica H. Schäf. and var. salmonea (Mout.) H. Schäf., and their close relatives V. johannis, V. hyaeniscyamus, V. serratifolia and V. kalakhensis have little to offer Mediterranean agriculture on the basis of poor agronomy.     

Vesicular-arbuscular mycorrhizae screened for Troyer citrange

Summary Troyer citrange is an important citrus root-stock but a slow grower. Eighteen different vesicular-arbuscular mycorrhizal (VAM) fungi were screened for their symbiotic response with Troyer citrange in unsterilized soil. Of the 18 fungi,Glomus macrocarpum (Ruakura),G. caledonicum (Nedlands),G. velum (Nedlands),G. monosporum (Nedlands),Gigaspora margarita (Ruakura), gave the greatest improvements in growth and nutrition, resulting in larger leaf area, plant height, stem diameter, and plant biomass, with higher P, Zn and Cu contents. All the five fungi were statistically on a par with each other in improving the growth and nutrition of Troyer citrange. Inoculations with these fungi allowed the rootstock to reach a stem diameter of pencil thickness, i.e., ready for budding, in about 13–14 months in contrast to 18–19 months without mycorrhizal inoculations, thus saving about 5–6 months.     

Pre-inoculation with arbuscular mycorrhizal fungi suppresses root knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

A pot experiment was conducted to evaluate the influence of pre-inoculation of cucumber plants with each of the three arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus mosseae, and Glomus versiforme on reproduction of the root knot nematode Meloidogyne incognita. All three AM fungi tested significantly reduced the root galling index, which is the percentage of total roots forming galls. Numbers of galls per root system were significantly reduced only in the G. intraradices + M. incognita treatment. The number of eggs per root system was significantly decreased by AM fungus inoculation, no significant difference among the three AM fungal isolates. AM inoculation substantially decreased the number of females, the number of eggs g⁻¹ root and of the number of eggs per egg mass. The number of egg masses g⁻¹ root was greatly reduced by inoculation with G. mosseae or G. versiforme. By considering plant growth, nutrient uptake, and the suppression of M. incognita together, G. mosseae and G. versiforme were more effective than G. intraradices.     

Infectivity and acetylene reduction of diazotrophic rhizosphere bacteria in wheat (Triticum aestivum) seedlings under gnotobiotic conditions

Summary Wheat seedlings were inoculated with rhizosphere nitrogen-fixing bacteria and grown gnotobiotically for 15 days. The growth medium consisted of semisolid agar with or without plant nutrients. The bacteria, isolated from roots of field-grown wheat, were three unidentified Gram-negative rods (A1, A2, E1), one Enterobacter agglomerans (C1) and two Bacillus polymyxa (B1, B2). A strain of Azospirillum brasilense (USA 10) was included for comparison.Nitrogenase activity (acetylene reduction activity, ARA) was tested on intact plants after 8 and 15 days of growth. In semisolid agar without plant nutrients, five isolates showed ARA of 0.01–0.9 nmol C₂H₄ plant^–1 h^–1, while the two strains of B. polymyxa had higher ARA of 3.3–10.6 nmol C₂H₄ plant^–1 h^–1.Plant development was not affected by inoculation with bacteria, except that inoculation with B. polymyxa resulted in shorter shoots and lower root weight.Transmission electronmicroscopy of roots revealed different degrees of infection. A. brasilense, A1 and A2, occurred mainly in the mucilage on the root surface and between outer epidermal cells (low infectivity). B. polymyxa strains and E1 were found in and between epidermal cells (intermediate infectivity) while E. agglomerans invaded the cortex and was occasionally found within the stele (high infectivity).     

Exposure and effects assessments of Bt-maize on non-target organisms (gastropods, microarthropods, mycorrhizal fungi) in microcosms

Potential differences between Bt-maize (MEB307 expressing the insecticidal Cry1Ab protein) and a near-isogenic non-Bt variety (Monumental) in their influence on the garden snail (Helix aspersa), soil microarthropods (Collembola, Actinedida, Acaridida, Gamasida and Oribatida) and mycorrhizal fungi were studied. Growing snails were caged in microcosms allowing the development of Bt or non-Bt-maize (Zea mays L.) on a sandy loam soil. After 3 months exposure, survival and growth of snails were similar in both treatments. Cry1Ab protein was detected in the Bt-maize leaves (22–42.2 μg Bt protein g⁻¹ dry wt), in the snail tissues (0.04–0.11 μg Bt-protein g⁻¹ dry wt) and in their faeces (0.034–5 μg Bt-protein g⁻¹ dry wt). Total soil microarthropod abundance and diversity were similar between control (non-Bt-maize) and the genetically modified (GM) Bt-maize microcosms. The mycorrhizal colonization of roots did not differ between Bt and non-Bt-maize (frequency of mycorrhizal roots was 88.7% and 83.3% respectively). The mycorrhizal infectivity of soils, expressed as MI₅₀ (minimum soil dry weight required to colonize 50% of plants) was measured using red clover. MI₅₀ was similar for soils where Bt or non-Bt-maize was cultivated for 4 months. The detection of Cry1Ab protein in the viscera and faeces of H. aspersa exposed to Bt-maize indicates that snails contribute to the transfer of the Bt-protein from plant to soil or snail predators. This may constitute an alternative route of exposure for Bt-protein in soil, but this was without a negative influence on mycorrhizal fungi or microarthropods. Results showed that Bt-maize was not toxic for the selected non-target species exposed for 3 or 4 months. The microcosms and analyses used in this study represent new methods for assessing effects of chronic exposure to GM plants of several diverse, yet ecologically and temporally associated species. As the soil organisms we studied can also be used in standardized ecotoxicological tests (XP X31-205-2 for mycorrhizal fungi, ISO 11267 for Collembola and ISO 15952 for snails), microcosm exposures represent a way to link laboratory and field methods for the ecotoxicological evaluation of GM plants.     

Selection of efficient vesicular-arbuscular mycorrhizal fungi for wetland rice (Oryza sativa L.)

Summary We tested the response of the wetland rice cultivar Prakash to inoculation with ten vescular-arbucular mycorrhizal (VAM) fungi (three selected from the first screening and seven isolated from local paddy fields) in a pot experiment under flooded conditions in order to select the most efficient mycorrhizal fungi to inoculate the rice nursery. A sandy clay loam soil was used as the substrate, fertilized with the recommended N and K levels (100 kg N ha^–1 as ammonium sulphate and 50 kg K ha^–1 as muriate of potash) and half the recommended level of P (25 kg ha^–1 as super phosphate). The inoculation was made into dry nursery beds and the beds were flooded when the seedlings were about 25 cm high, in 15 days. Twenty-eight-day old seedlings were transferred to pots filled with well puddled soil flooded with 5 cm of standing water. Based on the increase in grain yield and total biomass, Glomus intraradices and Acaulospora sp. were considered efficient and suitable for inoculation into rice nurseries.     

Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants

Plant growth-promoting rhizobacteria and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere. The role of different strains of Azospirillum on AM fungi development was evaluated by measuring the percentage of AM colonisation of the root system in durum wheat and maize plants, grown under both greenhouse and field conditions. The effect of wild-type Azospirillum brasilense strain Sp245 and genetically modified (GM) derivatives overproducing indole-3-acetic acid was assessed at greenhouse level in (1) three different cultivars of durum wheat, in the presence of indigenous AM fungi and (2) maize plants artificially inoculated with Glomus mosseae and Glomus macrocarpum. In addition, the establishment of natural AM fungal symbiosis was evaluated using Azospirillum lipoferum CRT1 in maize plants at field level. Despite the stimulatory effect of the different Azospirillum inocula on root growth, no significant differences in AM colonisation were found, independently of the AM fungus involved, either in wheat or in maize plants. Similarly, GM A. brasilense, which strongly stimulates root development, did not affect AM formation. Although these results were obtained in conditions in which the mycorrhization rate was moderate (15–30%), overall considered they indicate that the use of wild-type or GM Azospirillum phytostimulators does not alter mycorrhization.