Respiratory and photosynthetic C and N metabolism of nodulated Lupin roots during phosphorus deficiency

Philosophiae Doctor - PhDGrowth of symbiotic legume hosts is P limited, because of the high energetic requirements associated with N2 fixation. Attempts to overcome P deficiency in soils where legumes are grown involve addition of P-based fertilisers. However, these are produced from fmite, non-renewable resources that could be exhausted in the next 50-80 years. For this and other prudent reasons, viable alternatives are sought that include producing genetically enhanced plants with better P use efficiency (PUE). There exist some inter- and intraspecific genetic variation for associated traits of PUE in various legumes and these will have to be exploited to realize the development of P efficient cultivars. With the advent of sophisticated molecular tools, good progress has been made to understand the molecular response of some common physiological and morphological functions observed under LP. The research aims here were to investigate the energy costs and the alternative metabolic routes associated with C and N metabolism under LP in legumes, which is very scant in literature. We also investigated the recovery responses of nodulated roots upon P alleviation. Consequently, improvement strategies to produce legume varieties for better adaptation in poor P soils are envisaged. We have demonstrated varying degrees of sensitivity between the amide and ureide legume systems being investigated under short-term LP. The species-specific responses were ascribed to differences related to the agro-climatic origins, nodule morphologies and the type of N containing export product of the different legume types. These different responses also underscore possible different regulatory mechanisms under LP. Lupins were probed further, because of its apparent tolerance to P deficiency. Lupin nodules had between 3 to 5-fold higher Pj concentrations compared with soybeans under LP and HP, respectively. The maintenance of Pj levels, as oppose to a decline in the total P pool, is discussed in relation to its role in maintaining N2 fixation in lupins. Under LP, an effective Pj recycling mechanism in nodules is proposed to occur via the induction of the PEPc- MDH-ME route. This route also enhanced the capacity of root nodules to procure high malate concentrations that are used to fuel bacteroid respiration and N2 fixation. Two distinctly different cMDH proteins, one corresponding to HP and another corresponding to LP, were identified. The high malate concentrations reported here are speculated to have arisen through LP-induced cMDH. Metabolically available Pj decline developed gradually as P deficiency progressed. This coincided with a 15% decline in the %Ndfa. Moreover, under prolonged P deficiency the disproportionate synthesis of organic acids, most notably malate, that occurred at the expense of amino acids was proposed to account for this decline. The recovery in response to alleviation from LP involved alterations in the allocation of respiratory costs to growth and nutrient acquisition. Under LP, smaller nodules were formed and nodule metabolism revolved around accentuating PUE. Thus, there is considerable potential for improvement of P efficiency in legumes through manipulation of root: shoot partitioning

Phosphoenolpyruvate (PEP) metabolism in roots and nodules of Lupinus angustifolius under P stress

Thesis (MSc)--Stellenbosch University, 2004.ENGLISH ABSTRACT: This study investigated the activities of several of the enzymes involved in the alternative route of PEP metabolism via PEPc (EC 4.1.1.31). This reaction circumvents the adenyl ate-controlled PK (EC 2.7.1.40) reaction of the conventional glycolytic network under conditions of P stress. It was hypothesized that the synthesis of pyruvate under Pi stress would induce the PEPc alternative route and that C for pyruvate synthesis would primarily be imported via this route. This was assessed by looking at how total enzyme activities are perturbed under P stress and also by following the route of radioactive labelled 14C02 under sufficient (2 mM) and deficient P (2 JlM) conditions in either roots or nodules. The significance of the pathway under P stress, was further assessed by determining pool sizes of pyruvate that was synthesized from PEPc-derived C. The experiments were conducted under glasshouse conditions, as two separate studies: one to investigate the phenomenon of Pi stress and its consequences for PEPc-derived C metabolism, and the other one to study the enzymes involved. Seeds of Lupinus angustifolius (cv. Wonga) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 JlM P04 (LP) or 2 mM P04 (control) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. LP roots synthesized more pyruvate from malate than LP nodules, indicating the engagement of the PEPc route under Pi stress. In this regard, pyruvate is considered as a key metabolite under Pi stress. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the PK and PEPc routes. The enhanced PK activities supported these high pyruvate levels. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. The novelty of our results for symbiotic roots demonstrates that using metabolically available Pi is indeed a more sensitive indicator ofP stress. These results show that under Pi stress, nodules are able to maintain their Pi and adenylate levels, possibly at the expense of the root. It is suggested that nodules do not experience P stress to the same extent as roots or alternatively function optimally under conditions of low P availability. The increase in concentration of pyruvate synthesized from malate, indeed suggest that under LP conditions there is an increase requirement for pyruvate. It is clear from this data that the operation of bypass route in nodules should be investigated further. Nevertheless, this study provided incentives for understanding the role of C pathways in Ni-fixation, in particular under conditions ofP limitation.AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die aktiwiteite van verskeie ensieme van die alternatiewe metaboliese roete via phosphoenolpirovaat karboksilase (pEPc, EC 4.1.1.31) te ondersoek. Dié reaksie omseil die adenilaat-beheerde pirovaatkinase (PK, EC 2.7.1.40) reaksie van die konvensionele glikolitiese weg onder toestande van fosfaat (P) stremming. Dit is gepostuleer dat die sintese van pirovaat onder toestande van Pstremming die alternatiewe roete via PEPc sou induseer en dat die koolstof (C) vir pirovaatsintese gevolglik hoofsaaklik vanaf hierdie roete sou kom. Dit is bepaal deur die veranderinge in die totale ensiemaktiwiteite wat sou plaasvind onder P-stremming te ondersoek. Daar is ook gekyk na die roete' wat radioaktiewe C C4C02) sou volg in wortles en wortelknoppies wat behandel is deur blootsteling aan eerder lae fosfaat (2 1lM) of genoegsame fosfaat (2 mM; kontrole), Die betekenis van die alternatiewe roete is ook ondersoek deur die poel-groottes van pirovaat, soos gesintetiseer via die PEPc reaksie, te bepaal. Twee eksperimente is in 'n glashuis uitgevoer. Eerstens is die verskynsel van Pstremming, asook die invloed daarvan op PEPc-afgeleide C-metabolisme, bepaal. Tweedens is die betrokke ensieme bestudeer. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en in 'n waterkultuur gekweek. Die houers is voorsien met óf2 IlM P04 (LP) óf 2 mM P04 (HP) en lug wat 360 ppm C02 bevat het. Wortels, anders as wortelknoppies, het 'n betekenisvolle afname in anorganiese P (Pi) ervaar. Onder P-stremming, het lae fosfaat wortels meer pirovaat vanaf malaat gesintetiseer as wortelknoppies, wat 'n definitiewe bydrae vanaf die PEPc roete impliseer. Hiervolgens is pirovaat 'n sleutel metaboliet onder P-stremming. Die belangrikheid van die akkumulering van pirovaat onder P-stremmende toestande is verder beklemtoon deur die toename in metabolisme van PEP via beide die PK- en die PEPcreaksies. Die toename in PK-aktiwiteite is goed gekorreleer met die verhoogde produksie van pirovaat. Onder toestande van P-stremming het die aktiwiteit van PEPc in wortels verhoog, maar nie in wortelknoppies nie. Dit was nie die gevolg van 'n verhoogde uitdrukking van die ensiem nie. Wortel- en wortelknoppie- uitdrukking van PEPc is derhalwe nie gereguleer deur die uitdrukking daarvan nie, maar eerder deur post-tranlasie kontrole. Hierdie resultate vir wortels met wortelknoppies demonstreer dat metaboliese Pi 'n beter maatstaf is om P-stres aan te dui. Hierdie resultate toon dat wortelknoppies beter daartoe instaat is om hul Pi-vlakke en adenilaatvlakke te reguleer, en dit mag ten koste van die gasheerwortel wees. Ons stel voor dat wortelknoppies nie P-stremming tot dieselfde mate ervaar as die gasheerwortel nie en dat dié knoppies optimaal funksioneer by lae Pi vlakke. Die verhoogde konsentrasie van pirovaat, wat vanaf malaat gesintetiseer is, impliseer dat daar 'n groter vereiste is vir dié metaboliet onder toestande van Pstremming. Hierdie studie het die rol van koolstofmetabolismein stikstofbindende organismes, spesifiek onder toestande van fosfaat-tekort, beklemtoon

Co-detection of virulent escherichia coli genes in surface water sources

CITATION: Ndlovu, T., et al. 2015. Co-detection of virulent escherichia coli genes in surface water sources. PLoS ONE, 10(2):1-12, doi:10.1371/journal.pone.0116808.The original publication is available at http://journals.plos.org/plosoneMcNemar’s test and the Pearson Chi-square were used to assess the co-detection and observed frequency, respectively, for potentially virulent E. coli genes in river water. Conventional multiplex Polymerase Chain Reaction (PCR) assays confirmed the presence of the aggR gene (69%), ipaH gene (23%) and the stx gene (15%) carried by Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Enterohermorrhagic E. coli (EHEC), respectively, in river water samples collected from the Berg River (Paarl, South Africa). Only the aggR gene was present in 23% of samples collected from the Plankenburg River system (Stellenbosch, South Africa). In a comparative study, real-time multiplex PCR assays confirmed the presence of aggR (EAEC) in 69%, stx (EHEC) in 15%, ipaH (EIEC) in 31% and eae (EPEC) in 8% of the river water samples collected from the Berg River. In the Plankenburg River, aggR (EAEC) was detected in 46% of the samples, while eae (EPEC) was present in 15% of the water samples analyzed using real-time multiplex PCR in the Plankenburg River. Pearson Chi-square showed that there was no statistical difference (p > 0.05) between the conventional and real-time multiplex PCRs for the detection of virulent E. coli genes in water samples. However, the McNemar’s test showed some variation in the co-detection of virulent E. coli genes, for example, there was no statistical difference in the misclassification of the discordant results for stx versus ipaH, which implies that the ipaH gene was frequently detected with the stx gene. This study thus highlights the presence of virulent E. coli genes in river water and while early detection is crucial, quantitative microbial risk analysis has to be performed to identify and estimate the risk to human health.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116808Publisher's versio

Control strains of <i>E</i>. <i>coli</i> using virulent specific primers.

<p>Lane M: Generuler 1 kb plus (Fermentas); lane C: Multiplex of e<i>ae</i> (EPEC), <i>ipaH</i> (EIEC), <i>stx</i> (EHEC), <i>aggR</i> (EAEC); lane 1: <i>eae</i> (881 bp); lane 2: <i>ipaH</i> (619 bp); lane 3: <i>stx</i> (518 bp); lane 4: <i>aggR</i> (254 bp) and lane N: negative control.</p

High nitrogen rates do not increase canola yield and may affect soil bacterial functioning

CITATION: Becker, F., et al. 2019. High nitrogen rates do not increase canola yield and may affect soil bacterial functioning. Agronomy Journal, doi:10.2134/agronj2019.05.0359.The original publication is available at https://dl.sciencesocieties.org/publications/ajNitrogen fertilization has a fundamental role in agricultural productivity. However, injudicious N applications to crops are common. It is important to ensure the minimum N required for satisfactory crop growth is applied but that excess amounts are avoided due to potential impacts on agroecosystem functioning. Nitrogen at 0, 60, and 150 kg ha-1 was applied as limestone ammonium nitrate to plots arranged in a randomized complete block design, on three farms to determine the impact of rate and temporal distribution of fertilizer on canola (Brassica napus L.) production in South Africa, and the effect of N fertilizer application on the composition and diversity of soil bacterial communities. The amount and distribution of N had only minor effects on canola growth (P < 0.05) and no effects on yield or harvest index. Splitting fertilizer into two or three applications throughout the season resulted in more mineral N available in the soil later in the season. Increasing the N rate from 60 to 150 kg ha-1 had a significant impact on bacterial community composition. The lower rate favored bacteria that are more able to break down N-containing carbon sources. No effects of fertilizer amount or distribution were observed on either N fixation potential (number of nifH gene copies) or bacterial community diversity. Overall, a low rate of N fertilizer split into multiple applications is recommended for canola production, as higher rates do not increase yield and may have a detrimental impact on soil carbon and nitrogen cycling.https://dl.sciencesocieties.org/publications/aj/first-lookPost prin

DNA sequencing reveals high arbuscular mycorrhizal fungi diversity in the rhizosphere soil of Prunus africana trees in fragmented Afromontane forests

Abstract Purpose Arbuscular mycorrhizal fungi (AMF) play a key role in medicinal plant species, besides their ecological role in shaping plant communities. Knowledge about the diversity and structure of AMF communities associated with the endangered Prunus africana is valuable in the conservation and domestication of the species for its medicinal products. Methods We investigated the diversity and structure of AMF species communities in the rhizosphere soils of P. africana trees occurring in four fragmented Afromontane forests found in Cameroon (Mount Cameroon and Mount Manengouba) and Kenya (Chuka and Malava) using Illumina Miseq sequencing of 18S rRNA gene amplicons. Results A total of 64 virtual taxa (VT) belonging to eight genera were detected, namely Glomus (43 VT), Claroideoglomus (6 VT), Paraglomus (5 VT), Acaulospora (4 VT), Diversispora (3 VT), and Archaeospora, Pacispora, and Scutellospora with 1 VT each. Scutellospora heterogama VTX00286 was the most abundant and common species in all four sites (49.62%). Glomeraceae and Gigasporaceae were the most abundant families found across the sites, while Acaulosporaceae, Pacisporaceae, and Archaeosporaceae were rare, represented by < 1% of all the detected taxa. Conclusion Our data shows a high diversity of AMF species associated with P. africana and variable community structure partially shaped by local edaphic factors