Relationship of wild and cultivated forms of <Emphasis Type="Italic">Pisum</Emphasis> L. as inferred from an analysis of three markers,of the plastid,mitochondrial and nuclear genomes

Eighty-nine accessions of wild and cultivated peas (12 Pisum fulvum Sibth. et Smith., 7 P. abyssinicum A. Br., 31 wild and 42 cultivated forms of P. sativum L.) were analysed for presence of the variants of three functionally unrelated polymorphic markers referring to different cellular genomes. The plastid gene rbcL either contains or not the recognition site for restriction endonuclease AspLEI (rbcL+ vs. rbcL−); the mitochondrial gene cox1 either contains or not the recognition site for restriction endonuclease PsiI (cox1+ vs. cox1−); the nuclear encoded seed albumin SCA is represented by slow (SCA^S) or fast (SCA^F) variant. Most of the accessions possessed either of two marker combinations: 24 had SCA^F cox1+ rbcL+ (combination A) and 49 accessions had SCA^S cox1− rbcL− (combination B), 16 accessions represented 5 of the rest 6 possible combinations. All accessions of P. fulvum and P. abyssinicum had combination A, the overwhelming majority of cultivated forms of P. sativum had combination B while wild representatives of P. sativum had both combinations A and B, as well as rare combinations. This pattern indicates that combination A is the ancestral state in the genus Pisum L., inherited by P. fulvum and P. abyssinicum, while combination B seems to have arisen in some lineage of wild P. sativum which rapidly fixed mutational transitions of the three markers studied, most probably via a bottleneck effect during the Pleistocene. Then this ‘lineage B’ spread over Mediterranean and also gave rise to cultivated forms of P. sativum. Rare combinations may have resulted from occasional crosses between ‘lineage A’ and ‘lineage B’ in nature or during cultivation, or represent intermediate evolutionary lineages. The latter explanation seems relevant for an Egyptian cultivated form ‘Pisum jomardii Schrank’ (SCA^F cox1− rbcL−) which is here given a subspecies rank. Wild representatives of P. sativum could be subdivided in two subspecies corresponding to ‘lineage A’ and ‘lineage B’ but all available subspecies names seem to belong to lineage B only. Presently all wild forms would better be considered within a fuzzy paraphyletic subspecies P. sativum subsp. elatius (Bieb.) Schmalh. s. l.     

Polymorphism of <Emphasis Type="Italic">Got2</Emphasis> DNA sequences sheds light on <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss. intraspecies divergence and origin of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

DNA sequences of nuclear gene Got2 was studied in 60 accessions of Aegilops tauschii, 29 of subsp. tauschii and 31 of subsp. strangulata. It was found that Got2 allozyme polymorphism in Ae. tauschii is due to a single, unique, mutation which led to replacement of glutamic acid by isoleucine in residue 256 of the enzyme molecule, encoded by Got2. As revealed by Got2 DNA sequences variation, initially in its history Ae. tauschii was presented by subsp. strangulata, and among phylogenetic lineages of subsp. strangulata, the lineage “t-9¹s” (TauL3) is the most ancient, a relict one. Subspecies tauschii is relatively “young”. Initially it was presented by the lineage marked by combination of allozyme alleles Got2 ¹⁰⁵ and Acph1 ¹⁰⁰. In the past it inhabited the Continental area from Caucasia to Pakistan, but later on it was forced out by newly originated, now—a major lineage of subsp. tauschii, marked by Got2 ¹⁰⁰. This lineage extended the Continental area of the species up to Kirgizstan, but actually failed to penetrate into pre-Caspian area, occupied by subsp. strangulata. These results essentially differ from those obtained previously, using chloroplast DNA (cpDNA) sequences polymorphism. As revealed by cpDNA, the major, “usual”, subsp. strangulata (TauL2) is “younger” than subsp. tauschii, which resided on phylogenetic tree between relict lineage “t-9¹s”of subsp. strangulata—and major subsp. strangulata. But both cpDNA and Got2 DNA sequences indicate that the level of genetic variation in subsp. tauschii is much lower than in subsp. strangulata. According to Got2 DNA sequences variation, it was Ae. tauschii subsp. strangulata lineage “k-109″ which donated genome D to Triticum aestivum L. This lineage includes accessions: k-109 from South-Eastern Precaspian Azerbaijan; KU-2105, KU-2159 from Western Precaspian Iran; KU-2080 from Eastern Precaspian Iran.     

Effects of long-term mineral fertilization on microbial biomass,microbial activity,and the presence of <Emphasis Type="Italic">r</Emphasis>- and <Emphasis Type="Italic">K</Emphasis>-strategists in soil

Long-term effects of mineral fertilization on microbial biomass C (MBC), basal respiration (R _B), substrate-induced respiration (R _S), β-glucosidase activity, and the r–K-growth strategy of soil microflora were investigated using a field trial on grassland established in 1969. The experimental plots were fertilized at three rates of mineral N (0, 80, and 160 kg ha⁻¹ year⁻¹) with 32 kg P ha⁻¹ year⁻¹ and 100 kg K ha⁻¹ year⁻¹. No fertilizer was applied on the control plots (C). The application of a mineral fertilizer led to lower values of the MBC and R _B, probably as a result of fast mineralization of available substrate after an input of the mineral fertilizer. The application of mineral N decreased the content of C extracted by 0.5 M K₂SO₄ (C _ex). A positive correlation was found between pH and the proportion of active microflora (R _S/MBC). The specific growth rate (μ) of soil heterotrophs was higher in the fertilized than in unfertilized soils, suggesting the stimulation of r-strategists, probably as the result of the presence of available P and rhizodepositions. The cessation of fertilization with 320 kg N ha⁻¹ year⁻¹ (NF) in 1989 also stimulated r-strategists compared to C soil, probably as the result of the higher content of available P in the NF soil than in the C soil.     

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.     

Greenhouse evaluation of EDTA effectiveness at enhancing Cd,Cr, and Ni uptake in <Emphasis Type="Italic">Helianthus annuus</Emphasis> and <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Background, Aims and Scope  Phytoremediation is a promising means for the treatment of heavy metal contamination. Although several species have been identified as hyperaccumulators, most studies have been conducted with only one metal. Experiments were conducted to investigate the ability of Helianthus annuus and Thlaspi caerulescens to simultaneously uptake Cd, Cr and Ni. Materials and Methods  The efficiency of plants grown in a sandy-loam soil was investigated. The ability of two EDTA concentrations (0.1 and 0.3 g kg⁻¹) for enhancing the phytoremediation of Cd, Cr and Ni at two different metal concentrations (24.75 mg kg⁻¹ and 90 mg kg⁻¹) was studied. Results   Thlaspi hyperaccumulated Ni with 0.1 g kg⁻¹ EDTA. When the EDTA dosage was increased to 0.3 g kg⁻¹, Thlaspi was able to hyperaccumulate both Ni and Cr. Since Thlaspi is a low-biomass plant, it was considered insufficient for full-scale applications. Helianthus annuus hyperacummulated Cr (with 0.1 g kg⁻¹ EDTA) and Cd (0.3 g kg⁻¹ EDTA). Discussion  When the contamination was 8.25 mg kg⁻¹ per metal, the total metal uptake was 10–25% (1.35 to 2.12 mg) higher and had the same uptake selectivity (Cr>>Cd>Ni) for both EDTA levels. It was hypothesized that complexation with EDTA interfered with Ni translocation. For these experiments, the optimal results were obtained with the H. annuus-0.1 g kg⁻¹ EDTA combination. Conclusions  Although the use of EDTA did increase the amount of metal that could be extracted, care should be taken during in-situ field applications. Chelators can also increase the amount of metals that are leached past the root zone. Metal leaching and subsequent migration could lead to ground water contamination as well as lead to new soil contamination. Recommendations and Perspectives  Additional research to identify the optimal EDTA dosage for field applications is warranted. This is necessary to ensure that the metals do not leach past the root zone. Identification of a plant that can hyperaccumulate multiple metals is critical for phytoremediation to be a viable remediation alternative. In addition to being able to hyperaccumulate multiple metals, the optimal plant must be fast growing with sufficient biomass to sequester the heavy metals.     

DNA markers and pollen morphology reveal that <Emphasis Type="Italic">Praecitrullus fistulosus</Emphasis> is more closely related to <Emphasis Type="Italic">Benincasa hispida</Emphasis> than to <Emphasis Type="Italic">Citrullus</Emphasis> spp.

The round melon Praecitrullus fistulosus (Stocks) Pangalo has been cultivated in Asia since ancient times and has been considered an underexploited crop in the western world. In the USA, there is an increased interest in using P. fistulosus as a commercial vegetable, and possibly as a rootstock for grafting watermelon, melon, or cucumber. However, the taxonomic classification of P. fistulosus is incomplete and for many years it has been considered a close relative of watermelon [Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa] and was previously classified as Citrullus lanatus subsp. fistulosus (Stocks) Duthie et J.B. Fuller. Here, we used two sets of DNA markers to assess the genetic similarity of P. fistulosus in relation to Citrullus spp. {including Citrullus lanatus subsp. vulgaris, C. lanatus subsp. lanatus, Citroides group [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.], and C. colocynthis (L.) Schrad.}, Cucumis spp. (including C. melo, C. sativus, C. anguria, C. meeusei, C. zeyheri), Benincasa hispida (Thunb.) Cogn., Lagenaria siceraria (Mol.) Standl. and Cucurbita spp. (including C. moschata Duchesne and the winter squash C. maxima Duchesne). The first marker set comprised 501 markers that were produced by 38 primer pairs derived from watermelon expressed sequenced tags (ESTs) containing simple sequence repeat (SSR) motifs (designated as EST-SSR primers; produced 311 markers), and by 18 primer pairs derived from ESTs that do not contain SSR motives (designated here as EST-PCR primers; produced 190 markers). The second marker set comprised 628 markers that were produced by 18 sequence related amplified polymorphism (SRAP) primer pairs. The phylogenetic data indicated that among these cucurbit species, the wax gourd B. hispida is the closest to the P. fistulosus. Pollen observations, using light microscopy, indicated that each of the cucurbit genera examined here has unique pollen morphology. The Cucurbita spp. have globular pollen grains with a stigmatic surface. The L. siceraria has polygonal pollen grains with symmetrical boundaries, while the Citrullus spp. and Cucumis spp. have ovular (conical) and triangular shaped pollen grains (respectively). The B. hispida and P. fistulosus have spherical or semispherical pollen grains. These pollen features appear to be in agreement with the phylogenetic relationships of these two species based on DNA markers. Analysis with 12 SRAP primer pairs revealed low genetic diversity among 18 United States Plant Introductions (PIs) of P. fistulosus, indicating the need to expand the germplasm collection of this cucurbit crop.     

Molecular evidence of genetic diversity changes in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) germplasm after long-term maintenance

Maintaining germplasm genetic integrity is a key objective of long-term ex situ conservation. Periodic regeneration, performed on limited plots with small number of individuals, increases the risk of genetic drift and genetic diversity changes. In this study, six accessions of white flowered, dry seed pea varieties (Pisum sativum L. subsp. sativum var. sativum): Bohatyr, Klatovsky zeleny, Hanák, Moravsky hrotovicky krajovy, Raman and Viktoria-75 and four accessions of colour flowered, fodder pea (P. sativum subsp. sativum var. arvense (L.) Poiret: Arvika, Česky banán, Moravská krajová and Niké, representing Czechoslovak varieties and landraces, bred over the last 40–80 years, were analyzed using ten microsatellite locus specific markers. Each accession was represented by 20 individual seeds of two temporally different samples, spanning the period of 20 or 40 years. Together with intra-accession variation (except of cv. Hanák), evidence of genetic changes, e.g. differences in allele frequencies as well as genetic composition of sample, was detected in six out of ten accessions (Arvika, Bohatyr, Česky banán, Moravsky hrotovicky krajovy, Moravská krajová and Raman). Evidence of genetic erosion was found in three accessions (Česky banán, Moravsky hrotovicky krajovy and Raman), while in another three (Arvika, Bohatyr and Moravská krajová) the level of diversity was found to have increased. Moreover in three samples of Bohatyr (2004) and Klatovsky zeleny (1963 and 2004), low levels of heterozygosity was detected. These results demonstrate that in pea, a self-pollinating and highly homozygous plant, the danger of the loss of genetic integrity exists. These findings are significant for long-term ex situ germplasm management.     

Control of cotton <Emphasis Type="Italic">Verticillium</Emphasis> wilt and fungal diversity of rhizosphere soils by bio-organic fertilizer

Cotton Verticillium wilt is a destructive soil-borne disease affecting cotton production. In this study, application of bio-organic fertilizer (BIO) at the beginning of nursery growth and/or at the beginning of transplanting was evaluated for its ability to control Verticillium dahliae Kleb. The most efficient control of cotton Verticillium wilt was achieved when the nursery application of BIO was combined with a second application in transplanted soil, resulting in a wilt disease incidence of only 4.4%, compared with 90.0% in the control. Denaturing gradient gel electrophoresis patterns showed that the consecutive applications of BIO at nursery and transplanting stage resulted in the presence of a unique group of fungi not found in any other treatments. Humicola sp., Metarhizium anisopliae, and Chaetomium sp., which were considered to be beneficial fungi, were found in the BIO treatment, whereas some harmful fungi, such as Alternaria alternate, Coniochaeta velutina, and Chaetothyriales sp. were detected in the control. After the consecutive applications of BIO at nursery and transplanting stage, the V. dahliae population in the rhizosphere soil in the budding period, flowering and boll-forming stage, boll-opening stage, and at harvest time were 8.5 × 10², 3.1 × 10², 4.6 × 10², and 1.7 × 10² colony-forming units per gram of soil (cfu g⁻¹), respectively, which were significantly lower than in the control (6.1 × 10³, 3.4 × 10³, 5.2 × 10³, and 7.0 × 10³ cfu g⁻¹, respectively). These results indicate that the suggested application mode of BIO could effectively control cotton Verticillium wilt by significantly changing the fungal community structure and reducing the V. dahliae population in the rhizosphere soil.     

Production and cytogenetics of trigeneric hybrid involving <Emphasis Type="Italic">Triticum</Emphasis>, <Emphasis Type="Italic">Psathyrostachys</Emphasis> and <Emphasis Type="Italic">Secale</Emphasis>

Trigeneric hybrids may help establish evolutionary relationships among different genomes present in the same cellular-genetic background, and also offers the possibility to transfer different alien characters into cultivated wheat. In this study, a new trigeneric hybrid involving species from the Triticum, Psathyrostachys and Secale was synthesized by crossing wheat-P. huashanica amphiploid (PHW-SA) with wheat-S. cereale amphiploid (Zhongsi 828). The crossability of F₁ hybrid was high with 35.13%, and the fertility was 41.95%. The morphological characteristics of F₁ plants resembled the parent Zhongsi 828. The trigeneric hybrids pollen mother cells (PMCs) regularly revealed averagely 19.88 univalents, 9.63 ring bivalents, 3.97 rod bivalents, 0.60 trivalents and 0.03 tetravalents per cell. Multivalents consisted of trivalents and tetravalents can be observed in 52.7% of cells. A variation of abnormal lagging chromosome, micronuclei and chromosome bridge were formed at anaphase I and telophase II. The mean chromosomes number of F₂ progenies was 2n = 46.13, and the distribution range was 42–53. GISH results revealed that most F₂ plants had 6–12 S. cereale chromosomes, and only 0–2 P. huashanica chromosomes were detected. The results indicated that S. cereale chromosomes can be preferentially transmitted in the F₂ progenies of trigeneric hybrid than P. huashanica chromosomes. A survey of disease resistances revealed that the stripe rust resistance from the PHW-SA were completely expressed in the F₁ and some F₂ plants. The trigeneric hybrid could be a useful bridge for the transference of P. huashanica and S. cereale chromatins to common wheat.     

Divergent evolution of wild and cultivated subspecies of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> as revealed by the study of <Emphasis Type="Italic">PolA1</Emphasis> gene

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241–243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively.     

New data and phylogenetic placement of the enigmatic Old World lupin: <Emphasis Type="Italic">Lupinus mariae</Emphasis>-<Emphasis Type="Italic">josephi</Emphasis> H. Pascual

Lupinus mariae-josephi H. Pascual is an intriguing lupin species recently discovered in the Mediterranean region. New data from seed coat micromorphology, cytology, and DNA sequences were generated in order to extend our knowledge on this species and to examine its evolutionary relationships within Lupinus. This species shows morphological similarities with the Mediterranean smooth seeded species of sections Micranthi and Lutei. It shares the same chromosome number 2n = 52 with the latter Old World taxa, but also with unifoliolate lupins from Florida. Besides, L. mariae-josephi exhibited a seed coat micromorphology “intermediate” between the rough and the smooth seed coat types. Phylogenetic analyses using ITS and ETS nrDNA spacers, and the LEGCYC1A locus supported L. mariae-josephi as a distinct Old World line, placed out of the Scabrispermae, but without clear placement amongst the Mediterranean smooth-seeded lineages. Unexpectedly, LEGCYC1A data revealed phylogenetic affinities between L. mariae-josephi and L. villosus, a unifoliolate North American lupin that might have experienced a reticulated evolutionary process. All together, the data underline the phylogenetic interest of L. mariae-josephi in Lupinus and the need of additional investigations in order to definitely elucidate its enigmatic status. Moreover, as L. mariae-josephi is one of the rare Old World lupins strictly restricted to poor basic soils, it opens new perspectives of ecological and agronomic interests in the wide areas of poor calcareous soils in the Mediterranean region.     

Salt-tolerant rhizobacteria-mediated induced tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and chemical diversity in rhizosphere enhance plant growth

Salt-tolerant isolates Bacillus pumilus, Pseudomonas mendocina, Arthrobacter sp., Halomonas sp., and Nitrinicola lacisaponensis isolated from high saline habitats exhibited plant growth-promoting traits like P solubilization and indole acetic acid (IAA), siderophore, and ammonia production. These isolates were inoculated in wheat to assess microbe-mediated responses and plant growth promotion in salt affected soil. Maximum shoot and root length (33.8 and 13.6 cm) and shoot and root biomass (2.73 and 4.48 g dry weight) was recorded in plants inoculated with B. pumilus after 30 days. Total chlorophyll content was maximum in the leaves of the plants treated with Halomonas sp. (24.22 mg g⁻¹ dry weight) followed by B. pumilus (23.41 mg g⁻¹ dry weight) as compared to control (18.21 mg g⁻¹ dry weight) after 30 days. Total protein content was maximum in Arthrobacter sp. inoculated plant leaves (3.19 mg g⁻¹ dry weight) followed by B. pumilus (2.47 mg g⁻¹ dry weight) as compared to control (2.15 mg g⁻¹ dry weight) after 30 days. Total carotenoid content was maximum in plants inoculated with Halomonas sp. (1,075.45 and 1,113.29 μg g⁻¹ dry weight) in comparison to control (837.32 and 885.85 μg g⁻¹ dry weight) after 15 and 30 days. Inoculation of bacterial isolates increased presence of individual phenolics (gallic, caffeic, syringic, vanillic, ferulic, and cinnamic acids) and flavonoid quercetin in the rhizosphere soil. The concentration of IAA in rhizosphere soil and root exudates was also higher in all treatments than in control. Accumulation of phenolics and quercetin in the plants played a cumulative synergistic role that supported enhanced plant growth promotion of wheat in the stressed soil.     

Diversity analysis of <Emphasis Type="Italic">Aegilops</Emphasis> species from Morocco using RAPD markers

The diversity of 51 representative populations of the 5 Aegilops species from Moroccan collection was analyzed using 22 RAPD primers. We investigated the associations among these 5 Aegilops species (A. geniculata Roth (UUMM), A. triuncialis L. (UUCC), A. ventricosa Tausch (DDNN), A. peregrina (Hackel) Maire et Weiller (UUMM) and A. neglecta Req. ex Bert. subsp. recta (Zhuk.) K. Hammer (UUMMNN)); some diploid species considered as their ancestors; accessions of some neighboring countries and also accessions of Triticums. A total of 650 polymorphic RAPD fragments were amplified. A dendrogram was constructed using the un-weighed pair group method arithmetic average (UPGMA) and Jaccard`s similarity coefficient. The UPGMA clustering showed a regrouping to the level species with high level of the structuration of the diversity at A. geniculata. We confirm as reported by other authors, the proximity of N genome to U genome and C genome to M genome and also the difference between the genomes M and N. Thus, the phylogeny between the species and the different genomes were retracted.     

Characterization of the gibberellic acid response of the <Emphasis Type="Italic">Brassica napus</Emphasis> L. em. Metzg. dwarf mutant <Emphasis Type="Italic">NDF</Emphasis>-<Emphasis Type="Italic">1</Emphasis>

A novel dwarf mutant of Brassica napus L. em. Metzg., named NDF-1, was derived from a high doubled haploid line ‘3529’ of which seeds were jointly treated with chemical inducers and fast neutron bombardment. The germination results showed that the germination of NDF-1 was insensitive in response to exogenous gibberellic acid 3 (GA₃). The studies on growth response to exogenous GA₃ showed that NDF-1 seeding has at least 10-fold insensitivity than the wild-type. Moreover, no matter what concentrations of GA₃ were added to the seedlings and adult plants, the NDF-1 could not restore the wild type phenotype. These results indicated that the B. napus dwarf mutant NDF-1 was GA-insensitive mutant. The histological observations showed that the key reason of leading NDF-1 to dwarf was the reduction of hypocotyls and stems cell numbers.     

Performance of the Giant Reed (<Emphasis Type="Italic">Arundo donax</Emphasis>) in Experimental Wetlands Receiving Variable Loads of Industrial Stormwater

Two emergent macrophytes, Arundo donax and Phragmites australis, were established in experimental subsurface flow, gravel-based constructed wetlands (CWs) and challenged by untreated stormwater collected from the hard-pan and other surfaces of a dairy processing factory in south-west Victoria, Australia. The hydraulic loading rate was tested at two levels, sequentially, 3.75 and 7.5 cm day⁻¹. Some of the monitored variables were removed more efficiently by the planted beds in comparison to unplanted CWs (biochemical oxygen demand (BOD), total nitrogen (TN) and total phosphorus (TP); p < 0.007) but there was no significant difference between the A. donax and P. australis CWs in removal of BOD, suspended solids (SS) and TN (p > 0.007) at 3.75 cm day⁻¹ or SS and TN at 7.5 cm day⁻¹. At 3.75 cm day⁻¹, BOD, SS, TN and TP removal in the A. donax and P. australis CWs was 71%, 61%, 78% and 75% and 65%, 60%, 73% and 41%, respectively. Nutrient removal at 7.5 cm day⁻¹ in the A. donax and P. australis beds was 87%, 91%, 84% and 71% and 96%, 94%, 87% and 55%, respectively. As expected, the A. donax CWs produced considerably more biomass (10 ± 1.2 kg wet weight) than the P. australis CWs (2.7 ± 1.2 kg wet weight). This equates to approximately 107 and 36 tonnes ha⁻¹ year⁻¹ biomass (dry weight) for A. donax and P. australis, respectively (assuming 250 days of growing season and single-cut harvest). The performance similarity of the A. donax- and P. australis-planted CWs indicates that either may be used in HSSF wetlands treating dairy factory stormwater, although the planting of A. donax provides additional opportunities for secondary income streams through utilisation of the biomass produced.     

Assessing the Toxic Effects of Nickel,Cadmium and EDTA on Growth of the Plant Growth-Promoting Rhizobacterium <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis>

Plant growth-promoting rhizobacteria (PGPR) play an important role in the biodegradation of natural and xenobiotic organic compounds in soil. They can also alter heavy metal bioavailability and contribute to phytoremediation in the presence or absence of synthetic metal chelating agents. In this study, the inhibitory effect of Cd²⁺ and Ni²⁺ at different concentrations of Ca²⁺ and Mg²⁺, and the influence of the widely used chelator EDTA on growth of the PGPR Pseudomonas brassicacearum in a mineral salt medium with a mixture of four main plant exudates (glucose, fructose, citrate, succinate) was investigated. Therefore, the bacteriostatic effect of Cd²⁺, Ni²⁺ and EDTA on the maximum specific growth rate and the determination of EC50 values was used to quantify inhibitory impact. At high concentrations of Ca²⁺ (800 μmol L^-1) and Mg²⁺ (1,250 μmol L^-1), only a small inhibitory effect of Cd²⁺ and Ni²⁺ on growth of P. brassicacearum was observed (EC50 Cd²⁺, 18,849 ± 80 μmol L⁻¹; EC50 Ni²⁺, 3,578 ± 1,002 μmol L⁻¹). The inhibition was much greater at low concentrations of Ca²⁺ (25 μmol L⁻¹) and Mg²⁺ (100 μmol L⁻¹) (EC50 Cd²⁺, 85 ± 0.5 μmol L⁻¹ and EC Ni²⁺, 62 ± 1.8 μmol L⁻¹). For the chosen model system, a competitive effect of the ions Cd²⁺ and Ca²⁺ on the one hand and Ni²⁺ and Mg²⁺ on the other hand can be deduced. However, the toxicity of both, Cd²⁺ and Ni²⁺, could be significantly reduced by addition of EDTA, but if this chelating agent was added in stoichiometric excess to the cations, it also exhibited an inhibitory effect on growth of P. brassicacearum.     

A semi-fertile interspecific hybrid of <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. nigra</Emphasis> and the cytogenetic analysis of its progeny

Black rot is a bacterial disease of Brassica rapa caused by Xanthomonas campestris pv. campestris (Xcc.). Sources of resistance to this disease within B. rapa are insufficient and control measures are limited, making the development of resistant breeding lines extremely important. Certain lines of B. nigra exhibit very high resistance to Xcc. For this study, an interspecific cross between Brassica rapa and B. nigra was performed, and a total of 6 F₁ hybrids were obtained through ovary culture. Five plants (H_1–5) were relatively slow-growing, entirely sterile, and had 18 chromosomes in the majority of pollen mother cells (PMCs). GISH analysis showed that most of the PMCs had 8 B. nigra chromosomes, which indicated the expected AB genomic constitution. The last plant (H₆) was partially fertile and the majority of PMCs contained 10 chromosomes of B. rapa and 16 chromosomes of B. nigra, indicating an ABB genomic constitution. Ovary culture techniques were not necessary for the development of the first- or second-backcross generation. Sequence-related amplified polymorphism analysis of F₁, BC₁, and BC₂ plants indicated that some fragments from B. nigra were lost, particularly in the genome of BC₁/BC₂ in successive generation(s). The BC₂ plants expressing good resistance to Xcc. were observed.     

Identification of <Emphasis Type="Italic">Aegilops</Emphasis> L. species and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. based on chloroplast DNA

The genus Aegilops L. is a very important genetic resource for the breeding of bread wheat Triticum aestivum. Therefore, an accurate and easy identification of Aegilops species is required. Traditionally, identification of Aegilops species has relied heavily on morphological characters. These characters, however, are either not variable enough among Aegilops species or too plastic to be used for identification at the species level. Molecular markers that are more stable within species, therefore, could be the alternative strategy towards an accurate identification. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among 21 Aegilops species (including Ae. mutica that is now known as Amblyopyrum muticum) and between the latter and T. aestivum to generate markers for the diagnosis of all targeted species. Cleaved amplified polymorphic sequence (CAPS) applied on 22 coding and non-coding chloroplast regions using 80 endonucleases and sequencing of two of those regions revealed little polymorphism between T. aestivum and the various Aegilops species examined and to a less extent was the variation among Aegilops species. Polymorphism observed among species analysed allowed the discrimination of T. aestivum and 12 Aegilops species.     

N<Subscript>2</Subscript> fixation by faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) in a gypsum-amended sodic soil

The response of faba bean to the application of four rates of gypsum (0, 2.5, 5.0, 10.0 t ha⁻¹) to a non-saline, alkaline sodic soil was measured in terms of grain yield, dry matter (DM) production, N accumulation and the proportional dependence of the legume on symbiotic N₂ fixation (P _atm). A yield-independent, time-integrated ¹⁵N-dilution model was used to estimate symbiotic dependence. A significant decrease in the exchangeable sodium percentage and significant increases in exchangeable Ca⁺⁺ and the Ca⁺⁺:Mg⁺⁺ ratio in the 0–10-cm soil layer were measured 30 months after application of 10 t ha⁻¹ gypsum. Despite low and erratic rainfall during crop growth, faba bean DM and N uptake responded positively to gypsum application. The symbiotic dependence of the legume at physiological maturity was little affected by sodicity (P _atm = 0.74 at zero gypsum and 0.81–0.82 at 2.5–10 t ha⁻¹ gypsum). The increase in fixed N due to gypsum application was mainly due to increases in legume DM and total N uptake. At 10 t ha⁻¹ of gypsum, faba bean fixed more than 200 kg N ha⁻¹ in above-ground biomass.     

Morphological variation in <Emphasis Type="Italic">Laelia anceps</Emphasis> subsp. <Emphasis Type="Italic">dawsonii</Emphasis> f. <Emphasis Type="Italic">chilapensis</Emphasis> Soto-Arenas Orchidaceae in traditional home gardens of Chilapa,Guerrero, Mexico

Calaverita an endangered Mexican orchid is one of the most elegant and showy orchids of the genus Laelia and essential symbol within the local traditional celebrations of the Day of the Dead, in the region of Chilapa-Guerrero. We investigated morphological variation in flowers of la calaverita (Laelia anceps Lind. subsp. dawsonii (J. Anderson) Rolfe f. chilapensis Soto-Arenas, Orchidaceae) using the labellum of the specimens cultivated in traditional home gardens in the region of Chilapa, Guerrero, Mexico. The use of morphometric traits of the labellum is useful in appraising infra-subspecific morphological variation within the chilapensis form. Floral morphological variation exists among the specimens examined. These specimens, found exclusively in traditional home gardens, were grouped into four morphotypes: NAT, Topiltepec, Xulchuchío, and Terrero. The study of variation of la calaverita revealed the existence of four local morphotypes that originated from a long and complex process of selection of traits which responds to traditional preferences and knowledge about specific biotic and agroecological conditions. The cultural and aesthetic influence of traditional homegarden owners in the Chilapa region acts directly on selection, use and conservation of the morphological traits of la calaverita.