Spectroscopic Investigation of Tomato Seed Germination Stimulated by Trichoderma spp.

Seed germination is a complex process that can be negatively affected by numerous stresses. Trichoderma spp. are known as effective biocontrol agents as well as plant growth and germination stimulators. However, understanding of the early interactions between seeds and Trichoderma spp. remains limited. In the present paper, Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy were used to reveal the nature of tomato seed germination as stimulated by Trichoderma. A rapid response of tomato seeds to Trichoderma spp. was observed within 48 h on Murashige and Skoog medium (MS) substrate, preceding any physical contact. Raman analysis indicated that both Trichoderma species stimulated phenolic compound synthesis by triggering plant-specific responses in seed radicles. The impact of T. harzianum and T. brevicompactum on two tomato cultivars resulted in alterations to the middle lamella pectin, cellulose, and xyloglucan in the primary cell wall. The Raman spectra indicated increased xylan content in NA with T9 treatment as well as increased hemicelluloses in GZ with T4 treatment. Moreover, T4 treatment resulted in elevated conjugated aldehydes in lignin in GZ, whereas the trend was reversed in NA. Additionally, FTIR analysis revealed significant changes in total protein levels in Trichoderma spp.-treated tomato seed radicles, with simultaneous decreases in pectin and/or xyloglucan. Our results indicate that two complementary spectroscopic methods, FTIR and Raman spectroscopy, can give valuable information on rapid changes in the plant cell wall structure of tomato radicles during germination stimulated by Trichoderma spp.

Nutrients and soil structure influence furovirus infection of wheat.

Soil-borne wheat mosaic virus (SBWMV) and Soil-borne cereal mosaic virus (SBCMV), genus Furovirus, family Virgaviridae, cause significant crop losses in cereals. The viruses are transmitted by the soil-borne plasmodiophorid Polymyxa graminis. Inside P. graminis resting spores, the viruses persist in the soil for long time, which makes the disease difficult to combat. To open up novel possibilities for virus control, we explored the influence of physical and chemical soil properties on infection of wheat with SBWMV and SBCMV. Moreover, we investigated, whether infection rates are influenced by the nutritional state of the plants. Infection rates of susceptible wheat lines were correlated to soil structure parameters and nutrient contents in soil and plants. Our results show that SBWMV and SBCMV infection rates decrease the more water-impermeable the soil is and that virus transmission depends on pH. Moreover, we found that contents of several nutrients in the soil (e.g. phosphorous, magnesium, zinc) and in planta (e.g. nitrogen, carbon, boron, sulfur, calcium) affect SBWMV and SBCMV infection rates. The knowledge generated may help paving the way towards development of a microenvironment-adapted agriculture.

Screening of Native Trichoderma Species for Nickel and Copper Bioremediation Potential Determined by FTIR and XRF.

Soil pollution with heavy metals is a serious threat to the environment. However, soils polluted with heavy metals are considered good sources of native metal-resistant Trichoderma strains. Trichoderma spp. are free-living fungi commonly isolated from different ecosystems, establishing endophytic associations with plants. They have important ecological and biotechnological roles due to their production of a wide range of secondary metabolites, thus regulating plant growth and development or inducing resistance to plant pathogens. In this work we used indigenous Trichoderma strains that were previously isolated from different soil types to determine their tolerance to increased copper and nickel concentrations as well as mechanisms of metal removal. The concentrations of bioavailable metal concentrations were determined after extraction with diethylene-triamine pentaacetate (DTPA)-extractable metals (Cd, Cr, Co, Cu, Pb, Mn, Ni, and Zn) from the soil samples by inductively coupled plasma-optical emission spectrometry (ICP-OES). Two indigenous T. harzianum strains were selected for copper tolerance, and three indigenous T. longibrachiatum strains were selected for nickel tolerance tests. Strains were isolated from the soils with the highest and among the lowest DTPA-extractable metal concentrations to determine whether the adaptation to different concentrations of metals affects the mechanisms of remediation. Mechanisms of metal removal were determined using Fourier-transform infrared spectroscopy (FTIR) and X-ray fluorescence spectroscopy (XRF), non-destructive methods characterized by high measurement speed with little or no need for sample preparation and very low costs. Increased DTPA-extractable metal content for nickel and copper was detected in the soil samples above the target value (TV), and for nickel above the soil remediation intervention values (SRIVs), for total metal concentrations which were previously determined. The SRIV is a threshold of metal concentrations indicating a serious soil contamination, thus confirming the need for soil remediation. The use of FTIR and XRF methods revealed that the presence of both biosorption and accumulation of metals in the Trichoderma cells, providing good bioremediation potential for Ni and Cu.

Effects of Trichoderma harzianum on Photosynthetic Characteristics and Fruit Quality of Tomato Plants.

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gruzanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.

Threshold Level and Traceability of Roundup Ready® Soybeans in Tofu Production.

The aim of this study is to assess DNA degradation, DNA amplification, and GMO quantity during tofu production. Soybean seeds were spiked with Roundup Ready® soybeans (RRS) at 0.9, 2, 3 and 5% (by mass), to assess the level of RSS that would be of practical interest for threshold labelling. Real-time polymerase chain reaction (PCR) was more effective than conventional PCR in the analysis of raw soymilk, okara, boiled soymilk and tofu. The negative effect of grinding and mechanical manipulation was obvious in the okara sample prepared with 3 and 5% RRS, where GMO content was reduced to (2.28±0.23) and (2.74±0.26) %, respectively. However, heating at 100 °C for 10 min did not cause significant degradation of DNA in all samples. The content of RRS in the final product, tofu, was reduced tenfold during processing, ranging from 0.07 to 0.46%, which was below the labelling threshold level. The results are discussed in terms of global harmonization of GMO standards, which could have the positive effect on the trade of lightly processed foodstuffs such as tofu, especially regarding the labelling policies.

Effect of the edaphic factors and metal content in soil on the diversity of Trichoderma spp.

Influence of edaphic factors and metal content on diversity of Trichoderma species at 14 different soil sampling locations, on two depths, was examined. Forty-one Trichoderma isolates from 14 sampling sites were determined as nine species based on their internal transcribed spacer (ITS) sequences. Our results indicate that weakly alkaline soils are rich sources of Trichoderma strains. Also, higher contents of available K and P are connected with higher Trichoderma diversity. Increased metal content in soil was not inhibiting factor for Trichoderma species occurrence. Relationship between these factors was confirmed by locally weighted sequential smoothing (LOESS) nonparametric smoothing analysis. Trichoderma strain (Szeged Microbiology Collection (SZMC) 22669) from soil with concentrations of Cr and Ni above remediation values should be tested for its potential for bioremediation of these metals in polluted soils.

Zinc-induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall.

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido-reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²+. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H2 DCFDA and EPR measurements. The extent of zinc-induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H2O2 production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, •CH3 and •OH, were found in the cell walls of zinc-treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell-wall-bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc-promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²+ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.

Adjustment of diurnal starch turnover to short days: depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period.

A larger proportion of the fixed carbon is retained as starch in the leaf in short days, providing a larger store to support metabolism and carbon export during the long night. The mechanisms that facilitate this adjustment of the sink-source balance are unknown. Starchless pgm mutants were analysed to discover responses that are triggered when diurnal starch turnover is disturbed. Sugars accumulated to high levels during the day, and fell to very low levels by the middle of the night. Sugars rose rapidly in the roots and rosette after illumination, and decreased later in the light period. Global transcript profiling revealed only small differences between pgm and Col0 at the end of the day but large differences at the end of the night, when pgm resembled Col0 after a 4-6 h prolongation of the night and many genes required for biosynthesis and growth were repressed [Plant J. 37 (2004) 914]. It is concluded that transient sugar depletion at the end of the night inhibits carbon utilization at the start of the ensuing light period. A second set of experiments investigated the stimulation of starch synthesis in response to short days in wild-type Col0. In short days, sugars were very low in the roots and rosette at the end of the dark period, and after illumination accumulated rapidly in both organs to levels that were higher than in long days. The response resembles pgm, except that carbohydrate accumulated in the leaf as starch instead of sugars. A similar response was found after transfer from long to short days. Inclusion of sugar in the rooting medium attenuated the stimulation of starch synthesis. Post-translational activation of ADP-glucose pyrophosphorylase (AGPase) was increased in pgm, and in Col0 in short days. It is concluded that starch synthesis is stimulated in short day conditions because sugar depletion at the end of the night triggers a temporary inhibition of growth and carbohydrate utilization in the first part of the light period, leading to transient accumulation of sugar and activation of AGPase.