Dinitrogen fixation and infection of grass leaves byPseudomonas rubrisubalbicans andHerbaspirillum seropedicae

Bacteria causing mottled stripe disease in sugar cane, known asPseudomonas rubrisubalbicans, were shown to be able to fix molecular N₂ and to grow on it. The root associated diazotroph known asHerbaspirillum seropedicae, after artificial inoculation caused mottled stripe disease symptoms on sorghum and Napier grass but not on sugar cane. Both bacteria could be reisolated from leaves even 60 days after. Sugar cane leaves contained large numbers of these bacteria even in the uninoculated controls. Additional physiological characteristics of six strains ofP. rubrisubalbicans were compared with those of twoH. seropedicae strains and were shown to be very similar.     

Yield responses of Nepalese spring wheat (Triticum aestivum L.) cultivars to inoculation with Azospirillum spp. of Nepalese origin

Azosprilla were collected in wheat fields from subtropical and temperate soils of central Nepal at various elevations. Different wheat cultivars responded positively and significantly in grain yield, grain N-yield, and total N-yield in plant shoots to the inoculation with Nepalese isolate Azospirillum 10SW. Nepalese wheat cv. Seto responded significantly better with Azospirillum 10SW than with the Brasilian isolate A. lipoferum Sp 108 st, a strain which was found highly efficient in earlier experiments with German wheat cultivars, especially cv. Turbo. Yield of Turbo was increased by inoculations of both Azospirillum strains too, but it showed no significant differences depending from the inoculum used. The higher efficacy of combining Azospirillum 10SW and Seto, both collected from the same locality, indicates the possibility of improved associations using traditional cultivars and local bacteria. ei]{gnR O D}{fnDixon}     

Seed inoculation withAzospirillum brasilense andAzotobacter chroococcum and the root biomass of rice (Oryza sativa L.)

Summary The root biomass of rice seedlings increased due to inoculation withAzospirillum brasilense andAzotobacter chroococcum singly or in combination. In general, the increase in the biomass of roots was better in unsterilized soil than in sterilized one with or without inorganic nitrogen in the form of urea.     

Occurrence and survival ofAzospirillum spp. in temperate regions

In order to evaluate the suitability ofAzospirillum spp. as a crop inoculant in temperate regions, the natural occurrence, distribution and survival ofAzospirillum after seed inoculation in Belgian agricultural soils was studied.Azospirillum was present in most of the fields examined, but concentrations never exceeded 1000 cfu per g soil or per g roots. Under field conditions none of the known species was found to be localized inside the roots of barley, wheat, rye, maize or grasses. Also, the distribution ofA. brasilense SpBr 14 within the root system of hydroponic-grown wheat was studied by immunofluorescence. From the rhizosphere samples of the field crops investigated, a number of microaërophilic, diazotrophic bacteria were isolated and identified asA. lipoferum, found only on maize and grass roots, andA. brasilense, present under all crops. In contrast toA. brasilense, A. lipoferum was able to use different amino-acids and some derivatives as sole carbon and nitrogen sources. Use of a peat-based seed inoculant resulted in the establishment of theAzospirillum spp. in the rhizosphere of field-grown winter barley and winter wheat. The established population survived during winter without appreciable change in numbers, but there was no indication of active growth during spring or summer.     

Yield response of various wheat genotypes to inoculation withAzospirillum brasilense

Summary Twenty different wheat genotypes representing a wide genetic variability, were tested for their yield and yield components response to inoculation withAzospirillum brasilense, at two levels of N fertilization. Only two cultivars responded by a significant yield increase of 7.4 and 8.0 per cent — both at the higher N level. The response reflected an increase in the number of grains per plant added as additional spikes. The importance of the host plant genotype for a successful wheat-bacteria association is discussed.     

Response ofPanicum maximum Jacq. to inoculation withAzospirillum brasilense

Summary The effects on plant dry weight and acetylene reduction activity after applyingAzospirillum brasilense (strain 13t) to guineagrass,Panicum maximum Jacq., grown in clay pots under greenhouse conditions, are reported and discussed.     

Establishment of inoculatedAzospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

Summary Four field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

Indentification of Acetobacter diazotrophicus,Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans using biochemical and genetic criteria

Nitrogen-fixing Acetobacter diazotrophicus, Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans colonize sugar cane, and are thought to be capable of supplying high levels of fixed nitrogen to this plant. Eight A. diazotrophicus, two H. seropedicae and four H. rubrisubalbicans isolates were identified and compared by complementary biochemical and genetic methods. Utilization of carbon sources and antibiotic resistance patterns allowed differentiation of A. diazotrophicus from Herbaspirillum species. In order to distinguish strains within A. diazotrophicus species, the polymerase chain reaction was employed, using a Rhizobium meliloti dctA primer under low stringency hybridization conditions.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Nutrition of sorghum plants fertilized with nitrogen or inoculated withAzospirillum brasilense

Summary Sorghum plants were inoculated withAzospirillum brasilense or received an N-amended nutrient solution. Azospirillum inoculation increased plant dry weight and nitrogen assimilation by 25%. Most plant growth responses to Azospirillum were comparable to application of 2.0 mM N. Increased scavenging of nutrients, altered root permeability or nitrogen fixation are possible explanations for these effects.This work was supported by the United States Department of Agriculture, Agricultural Research Service (CRIS No. 5102-20170-001) in collaboration with the University of California, Berkeley. Requests for reqrints to G. J. Bethlenfalvay.     

Mechanisms of salt tolerance and interactive effects of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> inoculation on maize cultivars grown under salt stress conditions

The present work has been performed to study the growth and metabolic activities of two maize cultivars (cv. 323 and cv. 324) which are shown to have different tolerances to salt stress and to determine the effects of inoculation with Azospirillum spp. Along with identifying the mechanisms of maize salt tolerance and the role of Azospirillum (growth promoting rhizobacteria) in elevating salinity stress conditions is examined Maize cv. 323 was the most sensitive to salinity, while cultivar 324 was the most resistant of the 12 maize cultivars tested. Cultivars differences were apparent with certain growth criteria as well as related metabolic activities. The lack of a negative response to increasing NaCl concentration for water content, dry matter yield and leaf area of cv. 324 up to a concentration of – 0.6 MPa indicated salt tolerance. While for cv. 323 there was a marked inhibitory effect of salinity on growth. In the tolerant cv. 324, soluble and total saccharides, soluble protein in shoots and total protein in roots increased with salinity stress. The sensitivity of cv. 323 however was associated with depletion in saccharides and proteins. Proline accumulation was higher and detected earlier at a lower salinity concentration in the salt sensitive cv. 323 comapred to the salt tolerant cv. 324. When salt stressed maize was inoculated with Azospirillum, proline concentration declined significantly. The present study showed, in general, that the concentration of most amino acid increased on exposure to NaCl as well as when inoculated with Azospirillum. The relatively high salt tolerance of cv. 324, compared with cv. 323 was associated with a significantly high K⁺/Na⁺ ratio. Azospirillum inoculation markedly altered the selectivity of Na⁺, K⁺ and Ca⁺⁺ especially in the salt sensitive cultivar cv. 323. Azospirillum restricted Na⁺ uptake and enhanced the uptake of K⁺ and Ca⁺⁺ in cv. 323. A sharp reduction in the activity of nitrate reductase and nitrogenase in shoots and roots of both cultivars was induced by salinity stress. This reduction in NR and NA activity was highly significant at all salinity concentrations. Azospirillum inoculation stimulated NR and nitrogenase activity in both shoots and roots of both cultivars. The differential effect of Azospirillum inoculation on maize cv. 323 and cv. 324 illustrates the different sensitivity of these two cultivars to stress, but still does not provide any clues as to the key events leading to this difference.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2^nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.     

Evaluation and first-year field testing of efficient vesicular arbuscular mycorrhizal fungi for inoculation of wetland rice seedlings

Grain yields of the rice cultivar Prakash were improved upon inoculation with Glomus intraradices and G. fasciculatum, by 11% and 8%, respectively, compared with an uninoculated control. The results indicate that the amount of phosphate fertilizer usually applied to rice may be decreased by 50%, without affecting yield, if G. intraradices is inoculated.The authors are with the Department of Agricultural Microbiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560 065, India. ing author.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Occurrence ofAzospirillum spp. in coconut-based farming systems

Occurrence of Azospirillum was investigated in coconut-based farming systems, such as high-density multispecies cropping (15 crops), multi-storeyed cropping (3 crops), mixed cropping with tea and coffee (2 crops), intercropping with tropical tubers (5 crops), mixed farming with grasses (3 crops) and in 3 crops, arecanut,Mimosa invisa and sugarcane from other plots. A total of 26 plantation crops and intercrops were included in the study. Incidence of Azospirillum was determined by 2,3,5-triphenyl-tetrazolium chloride reduction and by culturing root fragments in N-free semisolid malate medium. Root samples from guava, mango and mimosa did not show any tetrazolium reduction or sub-surface pellicular growth. The extent of occurrence of Azospirillum seemed to depend upon the crop combinations. In a mixed farming system where guinea grass was one of the component crops, more root fragments of coconut and pepper demonstrated tetrazolium reduction activity than when guinea grass was absent.Azospirillum lipoferum andA. brasilense constituted 42% and 45% of the isolates, respectively, in the coconut-based cropping systems. Isolates from guinea grass, sugarcane and jackfruit exhibited higher nitrogenase (C₂H₂ reduction) than those isolated from plantation crops, tuber crops and spices. The large variation in the extent of association and nitrogenase activity of isolates from different crops indicated the need for inoculation with efficient cultures in a number of crops in coconut-based cropping systems.Contribution No. 613 of Central Plantation Crops Research Institute, Kasaragod.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

水稻种子内生泛菌(Pantoea spp.)系统发育多样性及其促生功能

【目的】Pantoea菌株是广泛分布在自然界中的一类功能多样的细菌。本研究对分离自水稻种子内生的Pantoea菌株进行系统发育分析及功能评价,从而确定分类地位、种类多样性、分布特征及功能特性。【方法】采用乙醇-次氯酸钠联合灭菌方法进行水稻种子的表面灭菌,进行内生细菌的分离与纯化;其次将纯化后的菌株进行16Sr RNA基因PCR扩增及序列分析,通过MEGA7软件构建系统发育树;将分离得到的菌株进行功能实验检测,如溶磷、产IAA、产铁载体、拮抗病原真菌等特性,最后检测菌株的溶血性;水稻分型采用SSR方法,并对水稻农学性状如分蘖数、株高、植株重及产量进行调查。【结果】本研究对分离自8个不同基因型水稻种子中的146株内生Pantoea菌株进行系统发育分析及功能评价,结果发现所分离到的泛菌菌株主要属于Pantoea dispersa、Pantoea agglomerans、Pantoea cypripedii以及Pantoea brenneri四个种,其中P. dispersa的菌株数量最多,分布最广,并且存在于所有的8个水稻种子样品中。对其中66株菌进行功能检测,发现86.3%和69.7%的菌株具有溶磷和产IAA能力,有7株菌具有产铁载体能力,未发现对真菌病害Fusarium moniliforme有拮抗作用的菌株,并发现3株菌具有溶血性;本实验未发现泛菌组成与水稻系统发育及农学性状存在明显的相关性。【结论】本研究首次对水稻种子中泛菌的多样性及其功能进行报道,发现不同基因型的水稻种子所含Pantoea种类及组成存在差异,种子选择性地积累了Pantoea类群,大部分菌株具有一定的促生特性。该研究结果有助于进一步探究微生物与植物的共进化、种子微生物的传播途径及作用方式。     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.