MgtE From Rhizobium leguminosarum Is a Mg2+ Channel Essential for Growth at Low pH and N2 Fixation on Specific Plants

MgtE is predicted to be a Rhizobium leguminosarum channel and is essential for growth when both Mg2+ is limiting and the pH is low. N2 was only fixed at 8% of the rate of wild type when the crop legume Pisum sativum was inoculated with an mgtE mutant of R. leguminosarum and, although bacteroids were present, they were few in number and not fully developed. R. leguminosarum MgtE was also essential for N2 fixation on the native legume Vicia hirsuta but not when in symbiosis with Vicia faba. The importance of MgtE and the relevance of the contrasting phenotypes is discussed.

Selected aspects of tiny vetch [Vicia hirsuta (L.) Gray S.F.] seed ecology: generative reproduction and effects of seed maturity and seed storage on seed germination

<i>Vicia hirsuta</i> (L.) Gray S.F. (tiny vetch) is a common and persistent segetal weed. Tiny vetch seeds and pods reach different stages of maturity during the crop harvest season. Some seeds that mature before cereal harvest are shed in the field and deposited in the soil seed bank, while others become incorporated into seed material. The objective of this study was to describe selected aspects of tiny vetch seed ecology: to determine the rate of individual reproduction of vetch plants growing in winter and spring grain crops and to evaluate the germination of seeds at different stages of maturity, subject to storage conditions. The seeds and pods of <i>V. hirsuta</i> were sorted according to their development stages at harvest and divided into two groups. The first group was stored under laboratory conditions for two months. In the autumn of the same year, the seeds were subjected to germination tests. The remaining seeds were stored in a storeroom, and were planted in soil in the spring. The germination rate was evaluated after 8 months of storage. Potential productivity (developed pods and flowers, fruit buds) was higher in plants fruiting in winter wheat than in spring barley. Vetch plants produced around 17-26% more pods (including cracked, mature, greenish-brown and green pods) and around 25% less buds in winter wheat than in spring barley. Immature seeds were characterized by the highest germination capacity. Following storage under laboratory conditions and stratification in soil, mature seeds germinated at a rate of several percent. After storage in a storeroom, seeds at all three development stages broke dormancy at a rate of 72- 75%. The high germination power of tiny vetch seeds stored in a storeroom indicates that this plant can be classified as an obligatory speirochoric weed species.

Effect of exogenous abscisic acid on accumulation of raffinose family oligosaccharides and galactosyl cyclitols in tiny vetch seeds (Vicia hirsuta [L.] S.F. Gray)

The role of the abscisic acid (ABA) in biosynthesis of raffinose family oligosaccharides (RFOs) and galactosyl cyclitols (Gal-C) in tiny vetch (<em>Vicia hirsuta</em> [L.] S.F. Gray) seeds was investigated. The ABA was applied through incubation of seed at various stage of its development. The level of RFOs and Gal-C was determined in seed maturing on plant and in seed maturing in vitro. In early stages of <em>V. hirsuta</em> seed development, the ABA activated the biosynthesis of galactinol, although the level of arisen galactinol quickly declined. In the later stages of <em>V. hirsuta</em> seed development ABA had stimulatory effect of RFOs and Gal-C biosynthesis. Influence of ABA on biosynthesis of a-galactosides in <em>Vicia hirsuta</em> seed seems to be dependent on abscisic acid concentration. Low concentration of ABA had stimulatory effect on a-galactosides biosynthesis, but high concentration of ABA inhibited the process.

Exogenously applied D-pinitol and D-chiro-inositol modifies the accumulation of α-D-galactosides in developing tiny vetch (Vicia hirsuta [L.] S.F. Gray) seeds

In the present study we have investigated the effect of exogenous cyclitols on the accumulation of their galactosides and raffinose family oligosaccharides (RFOs), as well as on some enzymes important for their biosynthesis in seeds of tiny vetch (<em>Vicia hirsuta</em> [L.] S.F. Gray). Immature seeds during 6-day incubation with D-<em>chiro</em>-inositol (naturally does not appear in seeds of tiny vetch) were accumulated cyclitol and its galactosides (fagopyritols: B1 and B2). Short 4-hour incubation with D-<em>chiro</em>-inositol, and subsequent slow desiccation process caused accumulation of free cyclitol only, without biosynthesis of its galactosides. Feeding D-<em>chiro</em>-inositol to pods of tiny vetch induced accumulation of high levels of its galactosides (fagopyritol B1, B2 and B3) in maturing seeds. Similarly, feeding D-pinitol increased accumulation of its mono-, di- and tri-galactosides: GPA, GPB, DGPA and TGPA in tiny vetch seed. Accumulation of both cyclitols and their galactosides drastically reduced accumulation of verbascose. Inhibition of RFOs biosynthesis by elevated levels of free cyclitols suggests some competition between formation of both types of galactosides and similarity of both biosynthetic routes in tiny vetch seeds. Galactinol synthase (GolS) from tiny vetch seeds demonstrated ability to utilize D-<em>chiro</em>-inositol as galactosyl acceptor, instead of myo-inositol. Presence of both cyclitols, as substrates for GolS, caused synthesis of their galactosides: fagopyritol B1 and galactinol. However, formation of galactinol was more efficient than fagopyritol B1. D-chiro-Inositol and D-pinitol at concentrations several-fold higher than myo-inositol had inhibitory effect on GolS. Thus, we suggest that a level of free cyclitols can have an influence on the rate of galactinol biosynthesis and further accumulation of RFOs and galactosyl cyclitols in tiny vetch seeds.

First Report of Ascochyta Blight of Vicia hirsuta (Hairy Tare) in the Republic of Georgia Caused by Ascochyta sp

Tan lesions with dark margins containing concentric rings of black pycnidia were observed on leaves and pods of hairy tare (Vicia hirsuta L.) growing near Ateni, GA (41°54.631′N, 44°05.586′E, elev. 730 m) on 1 July 2004. Lesions were reminiscent of those induced by Ascochyta rabiei (Pass.) Labrousse on chickpea (Cicer arietinum L.). At the time of collection, necrotic lesions were observed on the stems, leaflets, and pods of several plants. The fungus was isolated by surface-disinfecting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then deionized H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking cirrhi on 3% WA and picking germinated single conidia. After 14 days of growth, the isolated fungus had colony morphology similar to that of A. rabiei on V8 juice agar. A conidial suspension of the fungus (1 × 105 conidia/ml) was spray-inoculated onto 2-week-old plants including PI lines 628303, 628304, 420171, and 422499 of V. hirsuta and C. arietinum cv. Burpee. Plants were obtained from the USDA Western Region Plant Introduction Station, Pullman, WA, and 20 replicate plants of each genotype were inoculated. Inoculated plants were covered with a plastic cup to maintain high humidity and incubated in a growth chamber for 48 h at 18°C. Following removal of the cups, characteristic Ascochyta blight lesions were apparent 14 days after inoculation on both plant species. DNA was extracted from the isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of the chitin synthase 1 gene, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands and a BLAST search of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences revealed the chickpea pathogen Didymella rabiei (anamorph Ascochyta rabiei) accessions DQ383958, DQ386480, and DQ386488 as the closest matches in the databases with 95, 95, and 88% sequence similarity, respectively. These results, coupled with the morphological identification and the inoculation results, confirm the identity of the fungus as Ascochyta sp. Further research needs to be performed to determine if this represents a new species of Ascochyta. The identification of this fungus is part of a larger project to develop a phylogeny for Ascochyta spp. infecting cultivated legumes and their wild relatives that will provide a framework for the study of the evolution of host specificity and speciation of plant-pathogenic fungi. This is the second report of an Ascochyta species on V. hirsuta, and to our knowledge, the first report of Ascochyta blight of this host in the Republic of Georgia. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.