ABSTRACT Plant growth-promoting rhizobacterial (PGPR) strains R62 and R81 have previously been isolated and characterized as part of the Indo-Swiss Collaboration in Biotechnology. Here we present the draft genome sequences of these two PGPR strains, with the aim of unraveling the mechanisms behind their ability to promote wheat growth.

To promote conservation tillage in organic farming systems, weed control and ley removal within arable-ley rotations need to be optimized. A long-term field trial was thus established in Frick, Switzerland in 2002 on a clayey soil and with a mean precipitation of 1000 mm/year. The tillage experiment distinguished between conventional tillage with mouldboard ploughing (CT, 15 cm depth) and reduced tillage (RT), including a chisel plough (15 cm) and a stubble cleaner (5 cm). Results of a 2-year grass-clover ley (2006/2007) and silage maize (2008) are presented. Due to dry conditions, mean grass-clover yields were 25% higher in RT than in CT, indicating better water retention of RT soils. Clover cover and mineral contents of the fodder mixture were also higher in RT. The ley was successfully removed in autumn 2007 in RT plots, and a winter pea catch crop was sown before maize. In CT, ploughing took place in spring 2008. Maize yields were 34% higher in RT than in CT, despite a two- to three-fold higher but still tolerable weed infestation. Maize in RT plots benefited from an additional 61.5 kg of easily decomposable organic N/ha incorporated into the soil via the pea mulch. Measurement of arbuscular mycorrhizal colonization of maize roots indicated a similar mechanical disturbance of the topsoil through the reduced ley removal system compared with ploughing. It is suggested that RT is applicable in organic farming, even in arable-ley rotations, but long-term effects need further assessment.

Glomus spinuliferum forms globose to subglobose, dark yellow to orange brown spores in the soil and occasionally in roots of plants. Spores are 110-154 μm broad and have one wall consisting of 4 layers. The outer wall layer is hyaline and 1-2 pm thick. It may occasionally swell up to 50 μm in mounting media containing lactic acid. Wall layer 2 is very thin and hyaline and densely packed with spines up to 2 pm long which grow into wall layer 1. Wall layer 2 is tightly attached to layer 3, which is dark yellow to orange brown, laminated, and (6-)9-15 μm thick. The thin wall layer 4 is semi-flexible, hyaline to dark yellow and sometimes hard to see. The pore of the spore is often open but usually closed by wall layer 4 or by wall thickening of layer 3 and occasionally by a bridging septum originating from the laminated layer. The hyphal attachment is 8-15 μm broad, having several septae at some distance from the spore base. The new species is the second Glomalean fungus - after G. caesaris - that has been newly described from the nature reserve Kaiserstuhl in Germany.

Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G. max [L.] Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU). The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase. However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c. 6 mg x g(-1) dry weight up to 14 d, when nodules softened and decayed. The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules. These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.

A class III chitinase gene. Mtchitinase III-3, has been found to be induced in roots of Medicago truncatula specifically upon colonization by the arbuscular mycorrhizal fungi Glomus mosseae and Glomus intraradices. Here we show by in situ hybridization techniques that Mtchitinase III-3 is only expressed in cortical root cells containing arbuscules formed by either of the fungi. Transcripts of Mtchitinase III-3 were detected in cells containing developing as well as mature arbuscules. They were absent from cells adjacent to arbuscule-containing cells, from cells in contact with intraradical hyphae and from cortical cells in non-colonized areas of the root. These results indicate that the product of the Mtchitinase III-3 gene has a role within individual cortical cells during formation and functioning of arbuscules.

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.

Previous work has indicated that sugar sensing may be important in the regulation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fructosyltransferase (1-SST; EC 2.4.1.99) and Suc:fructan-6-fructosyltransferase (6-SFT; EC 2.4.1.10) were analyzed. Upon supply of Sue, fructan pool sizes increased markedly. Within 24 h, 1-SST activity was stimulated by a factor of three and 6-SFT-activity by a factor of more than 20, compared with control leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increased in the presence of the invertase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose stimulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc-alpha-1 and 1-alpha-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxylamine A, a potent trehalase inhibitor. Tre by itself, however, in the presence or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized, such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fructan synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-Man isomerase (EC 5.3.1.8) activity was detected. These results indicate that the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Sue, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.

Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

In Arabidopsis, genes encoding functional enzymes for the synthesis and degradation of trehalose have been detected recently. In this study we analyzed how trehalose affects the metabolism and development of Arabidopsis seedlings. Exogenously applied trehalose (25 mM) strongly reduced the elongation of the roots and, concomitantly, induced a strong accumulation of starch in the shoots, whereas the contents of soluble sugars were not increased. When Arabidopsis seedlings were grown on trehalose plus sucrose (Suc), root elongation was restored, but starch still accumulated to a much larger extent than during growth on Suc alone. The accumulation of starch in the shoots of trehalose-treated seedlings was accompanied by an increased activity of ADP-glucose pyrophosphorylase and an induction of the expression of the ADP-glucose pyrophosphorylase gene, ApL3. Even in the presence of 50 mM Suc, which itself also slightly induced ApL3, trehalose (5 mM) led to a further increase in ApL3 expression. These results suggest that trehalose interferes with carbon allocation to the sink tissues by inducing starch synthesis in the source tissues. Furthermore, trehalose induced the expression of the beta-amylase gene, AT-beta-Amy, in combination with Suc but not when trehalose was supplied alone, indicating that trehalose can modulate sugar-mediated gene expression.

Results from pot and microcosm studies in the greenhouse have shown that plant growth and foliar chemistry is altered by the presence and species composition of arbuscular mycorrhizal fungi (AMF). The growth and survival of herbivores which feed on plants could, as a consequence, also be affected by these mutualistic soil fungi. Consequently, interactions between AMF, plants and herbivores could occur. To test this, larvae of the common blue butterfly, Polyommatus icarus (Lycaenidae), were fed with sprigs of Lotus corniculatus (Fabaceae) plants which were inoculated with one of two different AMF species, with a mixture of these AMF species or with sprigs of plants which were not inoculated with AMF. Survival and larval weight of third instar larvae fed with plants colonised by AMF were greater than those of larvae fed with non-mycorrhizal plants. Survival of larvae feeding on non-mycorrhizal plants was 1.6 times lower than that of larvae feeding on plants inoculated with a mixture of AMF species and 3.8 times lower than that of larvae feeding on plants inoculated with single AMF species. Furthermore, larvae fed with non-mycorrhizal plants attained only about half the weight of larvae fed with mycorrhizal plants after 11 days of growth. These differences in larval performance might be explained by differences in leaf chemistry, since mycorrhizal plants had a 3 times higher leaf P concentration and a higher C/N-ratio. Our results, thus, show that the presence of belowground mutualistic soil fungi influences the performance of aboveground herbivores by altering their food quality. Larval consumption, larval food use and adult lipid concentrations of the common blue butterfly differed between larvae which were fed with plants inoculated with different AMF species. This suggests that the performance of herbivores is not only influenced by the presence of AMF but also depends on the identity of the AMF species colonising the host plants. Moreover, a significant interaction term between AMF species and maternal identity of the larvae occurred for adult dry weight, indicating that the performance of offspring from different females was differently influenced by AMF species composition. To our knowledge, these results show for the first time that the species composition of AMF communities can influence life-history traits of butterfly larvae and possibly herbivores in general.

The enzyme sucrose: sucrose 1‐fructosyltransferase was partially purified from barley leaf growth zones. Four steps (ammonium sulphate precipitation and polyethylene glycol precipitation, followed by chromatography on Concanavalin A‐sepharose and hydroxylapatite) yielded a 35‐fold purification. The resulting preparation of 1‐SST which still contained a number of different activities related to fructan metabolism, was subjected to preparative isoelectric focusing, and sections of the gel were analysed individually for 1‐SST and related activities, using sucrose and 1‐kestose as substrates. This procedure yielded a 196‐fold purification and revealed the presence of two isozymes of 1‐SST with pI values of 4.93 and 4.99, as determined by analytical isoelectric focusing of the corresponding fractions. Both isozymes produced glucose and 1‐kestose when incubated with sucrose. In addition, small amounts of 6‐kestose and tetrasaccharides were formed. In particular, one of the two 1‐SST isozymes yielded fructose when incubated with 1‐kestose, indicating that it also acts as a fructan exohydrolase. The other isozyme exhibited less fructan exohydrolase activity. Nystose was also degraded by the fructan exohydrolase activity but less than 1‐kestose, whereas 6‐kestose was not a substrate for the enzyme. Incubation of both 1‐SSTs with different concentrations of sucrose showed that the enzyme was not saturated even at 500 mM. As for the barley sucrose: fructan 6‐fructosyltransferase, both isozymes of 1‐SST yielded two polypeptide bands of molecular weight 50 and 22 kDa upon sodium dodecylsulphate polyacrylamide gel electrophoresis, suggesting their close relationship to invertase (composed of two subunits of similar size), as previously reported for other plants.

The effects of three conditions likely to affect soluble carbohydrate pools, namely drought, expression of barley sucrose: fructan 6‐fructosyl transferase (6‐SFT, EC 2.4.1.10) and the establishment of the arbuscular mycorrhizal symbiosis with Glomus mosseae were studied in a multifactorial experiment using tobacco (Nicotiana tabacum). Tobacco, a plant naturally unable to form fructan, accumulated fructan in leaves, and to a larger extent in the roots, when transformed with 6‐SFT. Under drought conditions, growth was considerably reduced, but neither expression of 6‐SFT nor mycorrhiza formation had an effect on growth rate. However, in response to drought, carbon partitioning was significantly altered towards accumulation of soluble sugars. In plants exposed to drought, pools of sucrose were greater than those of unstressed plants, particularly in their roots. In the transgenic plants expressing 6‐SFT, there were also increased contents of the products of 6‐SFT, namely fructan, most probably because of the increased availability of the substrate, sucrose. These effects were the same in the presence or absence of mycorrhiza. Hexoses (glucose and fructose) also increased in response to drought, primarily in the leaves. This effect of drought was little affected by the expression of 6‐SFT, except that it slightly enhanced drought‐induced glucose accumulation in roots. However, the presence of mycorrhiza led to a considerable reduction in drought‐induced accumulation of hexoses in the leaves. The content of the fungal disaccharide trehalose was greatly increased in the roots of all mycorrhizal plants upon exposure to drought, particularly in some of the transgenic plants expressing 6‐SFT.

ABSTRACT The TPS1 gene from Hansenula polymorpha , which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of TPS1 rendered H. polymorpha cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells’ ability to acquire thermotolerance.

The cDNA encoding sucrose‐fructan 6‐fructosyltransferase (6‐SFT) from barley ( Hordeum vulgare ) has been expressed in the methylotrophic yeast Pichia pastoris , using a translational fusion into vector pPICZαC, containing the N‐terminal signal sequence of Saccharomyces cerevisiae α‐factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6‐SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1‐SST activity when incubated with sucrose.

The effects of tomato and bean rhizospheres on hyphal spreading of the arbuscular mycorrhizal (AM) fungus Glomus mosseae were studied using a soil compartment system in combination with hydrophobic polytetrafluorethylene (PTFE) membranes. Both the nylon screen and the PTFE membrane were freely permeable to hyphae but not to roots. Furthermore, the hydrophobic PTFE membrane seemed to be a barrier to the flux of soil solutions containing root exudates. The results show that water soluble exudates of tomato and bean roots greatly stimulate hyphal growth in the soil compartment system used. Moreover, water soluble root exudates of bean exert a clear attractional effect on AM hyphal growth.

To study the response of non-mycorrhizal and mycorrhizal maize plants to drought, the changes in the pools of non-structural carbohydrates and amino acids were analysed in leaves and roots of two maize cvs. Plants well colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) (60% of root length infected) and comparable non-mycorrhizal plants were subjected to moderate drought stress by reducing the water supply. This stress induced a conspicuous increase in the trehalose pool in the mycorrhizal roots, probably because it was accumulated by the fungal symbiont. Furthermore, glucose and fructose were accumulated in leaves and roots of non-mycorrhizal plants but not in the mycorrhizal ones. Starch disappeared completely from the leaves of both mycorrhizal and non-mycorrhizal plants in response to drought. Activities of soluble acid invertase and trehalase were also measured. Acid invertase activity increased during drought in the leaves of both non-mycorrhizal and mycorrhizal plants whilst in the roots it was unaffected in non-mycorrhizal plants and decreased in the mycorrhizal ones. Without drought stress, trehalase activity was considerably higher in the leaves and roots of mycorrhizal plants than in those of non-mycorrhizal plants. It increased conspicuously during drought, primarily in the leaves of non-mycorrhizal plants. A drought-induced accumulation of amino acids as well as imino acids was found in roots and leaves of both mycorrhizal and non-mycorrhizal plants; leaves of mycorrhizal plants accumulated more imino acids than those of non-mycorrhizal ones. Our results show that drought stress and the presence of a mycorrhizal fungus have a considerable effect on carbon partitioning, imino acid and amino acid accumulation in maize plants.