Should emergence models for Lolium rigidum be changed throughout climatic conditions? The case of Spain

Lolium rigidum Gaudin is a troublesome weed worldwide, and its distribution may increase due to climate change. It has become resistant to several herbicides and, therefore, its control is problematic. On the other hand, its seed biology, with little dormancy and short-term viability, can play a major role in reducing its seed bank, which would improve control of this weed. For this reason, knowledge of the emergence patterns of this species entails improvements for its management. In this work seeds from one population of L. rigidum, collected in Northeastern Spain, were distributed and sown in 11 sites across Spain, and its emergence was followed every 2–7 days for two growing seasons (2016-17 and 2017–18). These emergence sequences were compared with previously published thermal time and hydrothermal time based models, and new models were developed afterwards. The hydrothermal time based model did not significantly improve models based solely on thermal time. The accuracy of these newly developed models varied from site to site, but in general, they worked better for Northern locations than for Southern locations. Thus, the experimental sites were split between North and South, and specific models for each region were developed. The model developed for Northern sites worked very well, but the one for Southern sites needs improvement. This works reveals that, with regard to this weed species, probably different models should be developed for different climatic conditions. The model developed with data from Northern sites should be validated with local populations, while the one developed with data from Southern sites will probably need to be revised or newly developed with data from more Southern populations.The group from the University of Lleida-Agrotecnio would also like to thank the Spanish Goverment for partial funding, through project AGL2017-83325-C4-2-R (AEI/FEDER/UE)

Nitrate reductase isozymes in Bradyrhizobium sp. (Lupinus) bacteroids: localisation, biochemical and kinetic characteristics

9 pages, figures, and tables statistics.Bradyrhizobium sp. (Lupinus) strains 750 and IM-43B, possessing nitrate reductase activity (NRA) were used to inoculate Lupinus albus cv. Multolupa plants. Bacteroids from root nodules were sonicated and sedimented to separate membranes from cytosolic fractions. Our results confirmed the existence of constitutive NRA mostly in the membranes, but soluble and membrane-bound NR forms were also induced by nitrate, and both of them used NADH as electron donor. Immunolocalisation of NR using a monoclonal antibody showed the presence of a larger number of epitopes in the soluble region than in membrane of bacteroids, and gold particles increased in those nodules grown with nitrate. Chromatographic purification of NR allowed the detection of two active fractions with an average MW of 91 and 362 kD. A further attempt to separate NR fractions in bacteroids and free-living cells grown with nitrate, led us to detect two more native fractions (180 and 720 kD). SDS-PAGE gel electrophoresis from all of the four native fractions revealed the presence of two common bands of around 20 and 70 kD; these results seem to indicate that all of the native enzymes were associations of one single ≅ 90 kD monomer molecule. Specific NRA increased from the small to the large proteins, suggesting positive cooperation by monomer associations. There was evidence enough to support an interpretation that constitutive and substrate-induced NRs would be formed by a monomeric and its homotetrameric isozyme. On the other hand, three IEF spots were obtained by 2-D gel electrophoresis and by anionic chromatography with pI 7.0, 7.8 and 8.1, when any of the native fractions were analysed. Kinetic studies of the four MW native isoforms showed that the two smallest molecules behaved according to Michaelis-Menten with similar KM, but the 360 and 720 kD fractions fitted much better to straight lines in a Hill plot, indicating allosterism and positive cooperativity.Peer reviewe

Isolation and characterisation of symbiotically effective Rhizobium resistant to arsenic and heavy metals after the toxic spill at the Aznalcóllar pyrite mine

After the toxic spill occurred at Aznalcóllar pyrite mine (Southern Spain), a wide area of croplands near the Doñana Wild Park was contaminated with 4.5 million m3 of slurries composed of acidic waters loaded with toxic metals and metalloids such as As, Sb, Zn, Pb, Cu, Co, Tl, Bi, Cd, Ag, Hg and Se. Today, 6 years after the spill, the concentration of toxic elements in these soils is still very high, in spite of the efforts to clean the zone. However, some plant species have colonised this contaminated area. Legumes possessing N2-fixing nodules on their roots represented a significant proportion of these plants. Our objective was to use the Rhizobium-legume symbiosis as a new tool for bioremediate the affected area. We have isolated about 100 Rhizobium strains, 41 of them being resistant to high concentrations of As (300 mg l-1), Cu (100 mg l-1) and Pb (500 mg l-1). Their phenotypes and bioaccumulation potentials have been characterised by their growth rates in media supplemented with As and heavy metals. The presence of the resistance genes in some strains has been confirmed by PCR and Southern blot hybridisation. Several Rhizobium were symbiotically effective in the contaminated soils. On the other hand, the first steps in nodule establishment seemed to be more affected by heavy metals than N 2-fixation.This research was financed by grant of the MCyT/INIA (Proyect RTA-02-015-C2-2). The authors are very grateful to Instituto de Formación Agraria y Pesquera de Andalucía (IFAPA) (formerly DGIA, Consejería de Agricultura y Pesca), Junta de Andalucía for their support. E.P., J.A.C. and P.A. are fellowship recipients of INIA (MCyT), Consejería de Agricultura y Pesca, Junta de Andalucía and Fundación Ramón Areces respectively.Peer Reviewe

Differential effects of coinoculations with Pseudomonas jessenii PS06 (a phosphate-solubilizing bacterium) and Mesorhizobium ciceri C-2/2 strains on the growth and seed yield of chickpea under greenhouse and field conditions.

First International Meeting on Microbial Phosphate Solubilization. Salamanca, Spain, 16-19 july 2002. 8 p.In the course of a project carried out in two regions of Spain, Castilla y Leo´ n and Andalucı´ a, aiming to find useful biofertilizers for staple grain-legumes, an efficient rhizobia nodulating chickpea (termed as C-2/2) and a powerful in vitro phosphate-solubilizing bacterial strain (termed as PS06) were isolated. Analyses of their 16S rDNA sequence indicated that they belong to the bacterial species Mesorhizobium ciceri and Pseudomonas jessenii, respectively. Greenhouse and field experiments were carried out in order to test the effect of single and dual inoculations on chickpea (ecotype ILC-482) growth. Under greenhouse conditions, plants inoculated with Mesorhizobium ciceri C-2/2 alone had the highest shoot dry weight. The inoculation treatment with P. jessenii PS06 yielded a shoot dry weight 14% greater than the uninoculated control treatment, but it was not correlated with shoot P contents. However, the co-inoculation of C-2/2 with PS06 resulted in a decrease in shoot dry weight with respect to the inoculation with C-2/2 alone. Under field conditions, plants inoculated with M. ciceri C-2/2, in single or dual inoculation, produced higher nodule fresh weight, nodule number and shoot N content than the other treatments. Inoculation with P. jessenii PS06 had no significant effect on plant growth. However, the co-inoculation treatment ranked the highest in seed yield (52% greater than the uninoculated control treatment) and nodule fresh weight. These data suggest that P. jessenii PS06 can act synergistically with M. ciceri C-2/2 in promoting chickpea growth. The contrasting results obtained between greenhouse and field experiments are discussed.This work was supported by the Junta de Castilla y Leo´ n and the Junta de Andalucı´ a. The authors thank the soil analyses service staff from IRNA for their help in this work.Peer reviewe

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus albus. I. N2 fixation potential Influência dos diferentes níveis de nitrato e estágio de crescimento em mutantes hipernodulantes de Lupinus albus I. potencial de fixação de N2

This work aimed to evaluate physiological parameters, nodulation response and N2 fixation rate in mutants of Lupinus albus in comparison with the standard Multolupa cultivar. Two nitrate levels (0 and 5mM) and two evaluation periods (7 and 10 weeks) were used. Significant differences were observed among genotypes, in relation to fresh nodule weight, nitrate levels and growth stages. The overall average for nitrate level differed between them where 5mM severely inhibited the number of nodules, reaching a 49.5% reduction in relation to treatment without nitrate. There were no behaviour differences among genotypes, nor among evaluation periods. Although the level of nitrate did not influence the production of shoot dry matter in relation to the average among levels applied, the L-135 genotype, being an inefficient mutant, reached very low values. There were no significant differences in electron allocation coefficient (EAC) among nitrate levels, nor among genotypes studied. However, the evaluation periods revealed differences, where the EAC for the seventh week had a higher value than that for the tenth week, when a 5mM aplication was evaluated. The N2 fixation rate (N2 FIX) showed the existence of the nitrate interference in fixation, given that the application of 5mM severely reduced. However, there were no differences among the genotypes and it was noted that the fixation rate was much higher in those that received nitrate. The L-88 and L-62 genotypes were the ones that have shown best adaptability in this experiment, thus being able to be recommended for new studies with higher nitrate levels and different evaluation periods. The nitrate (5mM) interferes in the nitrogen fixation rate, given that all the genotypes were affected by the level applied.<br>Este trabalho teve como objetivo avaliar parâmetros fisiológicos, resposta da nodulação e a taxa de fixação de N2 em mutantes de Lupinus albus comparando com a cultivar padrão Multolupa. Foram utilizados dois níveis de nitrato (0 e 5mM) e dois diferentes períodos de avaliação (7 e 10 semanas). No peso fresco dos nódulos, diferenças significativas entre genótipos em relação aos níveis de nitrato e estágios de crescimento foram observadas. Nível de nitrato de 5mM inibiu fortemente a produção de nódulos, chegando a ter uma redução de 49,5% em relação ao tratamento onde não se adicionou o nitrato. Em relação aos genótipos, os desempenhos não diferiram entre si, o mesmo acontecendo com os períodos avaliados. O nível de nitrato não influenciou na produção de matéria seca da parte aérea em relação à média entre os níveis aplicados. Entretanto, entre os genótipos existiu diferença, onde 0mM, os genótipos L-88 e L-62 apresentaram 6,7 e 6,4g, respectivamente. O genótipo L-135, por ser um mutante ineficiente alcançou valores extremamente baixos de matéria seca da parte aérea. Para o coeficiente de alocação de elétrons (EAC), não houve diferença significativa entre os níveis de nitrato, nem entre os genótipos estudados. Em compensação os períodos avaliados tiveram diferenças: na sétima semana a EAC apresentou valor superior à EAC na décima semana, quando foi avaliada na aplicação de 5mM. A taxa de fixação de N2 (N2 FIX) mostrou que existe interferência do nitrato na fixação, uma vez que, a aplicação de 5mM reduziu muito a fixação, apesar de que entre os genótipos não houve diferença entre si. Com estes resultados observamos que os genótipos L-88 e L-62 foram os que melhor se adaptaram podendo desta forma ser recomendados para novos estudos com maiores níveis de nitrato aplicados e diferentes períodos de avaliação. De uma forma geral ficou nítido que o nitrato (5mM) interfere na taxa de fixação de N2, uma vez que, todos os genótipos foram afetados pelo nível aplicado

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus albus. II. Enzymatic activity and transport of N in the xylem sap Diferentes níveis de nitrato e estágio de crescimento em mutantes hipernodulantes de Lupinus albus II. Atividade enzimática e transporte de N na seiva do xilema

The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by which the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS) and nitrogen compounds derived from fixation, such as N-<FONT FACE="Symbol">a</font>-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme activity was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate- resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nod- fix-) showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.<br>O estudo enzimático e o transporte de N na seiva do xilema foi realizado visando observar a influência de diferentes níveis de nitrato e estágios de crescimento da planta em mutantes tratadas quimicamente. Vários estresses induzem a redução no crescimento da planta da qual resulta na acumulação de aminoácidos livres, amidas ou ureídos, tanto na parte aérea como nas raízes e nódulos. A atividade de enzimas é decisiva em evitar produtos inibidores da nitrogenase pelo amônio, porém poucas são as informações deste mecanismo com o qual o xilema carrega estes produtos, em compensação, este processo, pode ser a chave da função em evitar acumulação de aminoácidos nas células dos nódulos infectados. O comportamento das enzimas nitrato redutase (NR), fosfoenolpiruvato carboxilase (PEPC), glutamina sintetase (GS) e dos compostos nitrogenados derivados da fixação como: N-<FONT FACE="Symbol">a</font>-amino, N-ureídos e N-amida em genótipos mutados foi observado. A atividade da enzima nitrato redutase foi altamente inflenciada pela aplicação de nitrato apresentando valores bastante superiores em relação a não aplicação do nitrato, independentemente dos genótipos, sendo que o melhor período de avaliação foi na décima semana. O genótipo L-62, com caracteríticas de resistência ao nitrato, mostrou de forma bem evidenciada que a enzima PEPC é inibida pela presença de nitrato. O genótipo L-135 (nod- fix-) apresentou atividade da GS extremamente baixo, mostrando desta forma que a GS é uma enzima altamente correlacionada com a fixação. Em relação ao melhor estágio de crescimento para a GS, o Lupinus albus, deve ser avaliado na sétima semana