Data_Sheet_1.ZIP

Apple replant disease (ARD) is a serious threat to producers of apple trees and fruits worldwide. The ARD etiology is not unraveled and managing options are either economically not applicable or environmentally harmful. Thus, interest is given in biomarkers that allow to indicate ARD situations at early time points in order to classify soils according to ARD severity but also to analyze the effectiveness to potential countermeasures. This study aimed at (i) identifying ARD biomarkers on the transcriptional level in root tissue by analyzing the expression of previously identified candidate genes in ARD soils of different origin and texture and (ii) testing the specificity of these marker genes to ARD. In vitro propagated M26 plantlets were submitted to a bio-test with three ARD soils, either untreated or disinfected by γ-irradiation. Expression of seven candidate genes identified in a previous transcriptomic study was investigated by RT-qPCR in a time course experiment. Already three days after planting, a prominent upregulation of the phytoalexin biosynthesis genes biphenyl synthase 3 (BIS3) and biphenyl 4-hydroxylase (B4Hb) was observed in the untreated ARD variants of all three soils. The phytoalexin composition in roots was comparable for all three soils and the total phytoalexin content correlated with the expression of BIS3 and B4Hb. The third promising candidate gene that was upregulated under ARD conditions was the ethylene-responsive transcription factor 1B-like (ERF1B). In a second experiment M26 plantlets were exposed to different abiotic stressors, namely heat, salt and nutrient starvation, and candidate gene expression was determined in the roots. The expression levels of BIS3 and B4Hb were highly and specifically upregulated in ARD soil, but not upon the abiotic stress conditions, whereas ERF1B also showed higher expression under heat stress. In conclusion, BIS3 and B4Hb are recommended as early ARD biomarkers due to their high expression levels and their high specificity.

Introduction Apple replant disease (ARD) causes reduced growth and fruit yield and affects orchards and tree nurseries worldwide. A number of pathogens have been consistently identified as causal agents of ARD; however factors affecting disease-severity are not fully understood. Aims We examined five soils from German tree nurseries and apple orchards featuring different soil characteristics and replant histories. We aimed to link the plant-soil interaction to replant disease severity. Methods In a greenhouse experiment, young apple plants were grown for eight weeks on untreated and disinfected (control) soils. Growth parameters were recorded to evaluate the severity of ARD. The defence response of the plants was examined by expression analysis of ARD indicator genes (BIS3, B4H and ERF1B) and GC–MS-based detection of phytoalexins. The fungal and bacterial rhizosphere communities were investigated by ITS and 16S rRNA amplicon sequencing, respectively. Results After eight weeks, ARD symptoms were observed on all soils. Growth depression was highest on soils that had faced intensive apple cultivation and lowest on a soil with only one year of apple cultivation prior to the experiment. These results correlated with increases in the BIS3 expression level and the phytoalexin content in the roots. No bacteria and fungi commonly found in increased abundance in ARD soils were consistently detected in all soils. Conclusions Replant history influenced disease severity more than soil characteristics. ARD symptoms correlated with BIS3 expression and phytoalexin (PA) formation. PA exudation increased the relative abundance of bacterial genera with the potential ability to degrade phenolic compounds.

Identification and expression profiles of putative leaf growth related microRNAs in maize (Zea mays L.) hybrid ADA313

Apple seedlings (ev. Melntosh were stunted when grown in pasteurized field soil to which four fungi, previously isolated from apple replant disease (ARD) soil, were added. Twenty‐one isolates of Bacillus sulnths produced inhibition zones in vitro against 20 ARD fungi. Isolate BACT‐1 ol B. subtilis showed significant antagonism against 17 fungi isolated from ARD soil on potato dexirose agar and apple plant height was increased in its presence in pesteurized and fertilized soil in glasshouse pot tests. Plant height also increased in the presence of isolate F.BW‐4 of B.subtilis.in pasteurized and fertilized soil in glasshouse pot tests but EBW‐4 liid not show antagonism against most of the ARD tungi in vitro. The growth of apple seedlings was stunted in the presence of 12 isolates of B. subtilis. However, fertilization of non pasteurized soil with NPK 1l‐55‐0 reversed the stunting effect. Under field conditions, BACT‐1 and EBW‐4 isolates. led to increased shoot growth in unfertilized and unppasteurized ARD soil.

(1) Background: The cultivation of resistant rootstocks is an effective way to prevent ARD. (2) Methods: 12-2 (self-named), T337, and M26 were planted in replanted and sterilized soil. The aboveground physiological indices were determined. (3) Results: The plant heights and the stem thicknesses of T337 and M26 were significantly affected by ARD. Relative chlorophyll content (June–October), Pn (August–September), and Gs (August) of T337 and relative chlorophyll content (June–July, September), Pn (September–October), and Ci (September) of M26 were significantly affected by ARD. ARD had a significant effect on Fv/Fm (June), qP (June–July), and NPQ of T337 (June–October, except August) and Fv/Fm (June) and NPQ (June-October, except July) of M26. Additionally, ARD affected Rfd of M26 and T337 during August. SOD (August and October), POD (August–September), and CAT (July-August, October) activities and MDA (September–October) content of T338 as well as SOD (July–October), POD (June–October), and CAT (July-October) activities and MDA (July, September–October) content of M26 were significantly affected by ARD. ARD significantly reduced nitrogen (October), phosphorus (September–October), and zinc (July) contents of M26 and potassium (June) content of T337. The above physiological indices were not affected by ARD in 12-2. (4) Conclusions: 12-2 could be useful as an important rootstock to relieve ARD due to strong resistance.

Mixed application of raw amino acid powder and Trichoderma harzianum fertilizer for the prevention and management of apple replant disease

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.

Arabidopsis plants responding to phosphorus (P) deficiency increase lateral root formation and reduce primary root elongation. In addition the number and length of root hairs increases in response to P deficiency. Here we studied the patterns of radical oxygen species (ROS) in the roots of Arabidopsis seedlings cultured on media supplemented with high or low P concentration. We found that P availability affected ROS distribution in the apical part of roots. If plants were grown on high P medium, ROS were located in the root elongation zone and quiescent centre. At low P ROS were absent in the elongation zone, however, their synthesis was detected in the primary root meristem. The proximal part of roots was characterized by ROS production in the lateral root primordia and in elongation zones of young lateral roots irrespective of P concentration in the medium. On the other hand, plants grown at high or low P differed in the pattern of ROS distribution in older lateral roots. At high P, the elongation zone was the primary site of ROS production. At low P, ROS were not detected in the elongation zone. However, they were present in the proximal part of the lateral root meristem. These results suggest that P deficiency affects ROS distribution in distal parts of Arabidopsis roots. Under P-sufficiency ROS maximum was observed in the elongation zone, under low P, ROS were not synthesized in this segment of the root, however, they were detected in the apical root meristem.

The overall survival of a plant depends on the development, growth, and functioning of the roots. Root development and growth are not only genetically programmed but are constantly influenced by environmental factors, with the roots adapting to such changes. The peptide AEDL (alanine–glutamine acid–asparagine acid–leucine) at a concentration of 10−7 M had an elongating effect on the root cells of Nicotiana tabacum seedlings. The action of this peptide at such a low concentration is similar to that of peptide phytohormones. In the presence of 150 mM NaCl, a strong distortion in the development and architecture of the tobacco roots was observed. However, the combined presence of AEDL and NaCl resulted in normal root development. In the presence of AEDL, reactive oxygen species (ROS) were detected in the elongation and root hair zones of the roots. The ROS marker fluorescence intensity in plant cells grown with AEDL was much lower than that of plant cells grown without the peptide. Thus, AEDL protected the root tissue from damage by oxidative stress caused by the toxic effects of NaCl. Localization and accumulation of AEDL at the root were tissue-specific. Fluorescence microscopy showed that FITC-AEDL predominantly localized in the zones of elongation and root hairs, with insignificant localization in the meristem zone. AEDL induced a change in the structural organization of chromatin. Structural changes in chromatin caused significant changes in the expression of numerous genes associated with the development and differentiation of the root system. In the roots of tobacco seedlings grown in the presence of AEDL, the expression of WOX family genes decreased, and differentiation of stem cells increased, which led to root elongation. However, in the presence of NaCl, elongation of the tobacco root occurred via a different mechanism involving genes of the expansin family that weaken the cell wall in the elongation zone. Root elongation of plants is of fundamental importance in biology and is especially relevant to crop production as it can affect crop yields.

Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N(1)-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N(1)-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root.

ABSTRACTThe Arabidopsis thaliana (hereafter Arabidopsis) root has become a useful model for studying how organ morphogenesis emerge from the coordination and balance of cell proliferation and differentiation, as both processes may be observed and quantified in the root at different stages of development. Hence, being able to objectively identify and delimit the different stages of root development has been very important. Up to now, three different zones along the longitudinal axis of the primary root of Arabidopsis, have been identified: the root apical meristematic zone (RAM) with two domains [the proliferative (PD) and the transition domain (TD)], the elongation zone (EZ) and the maturation zone (MZ). We previously reported a method to quantify the length of the cells of the meristematic and the elongation zone, as well as the boundaries or transitions between the root domains along the growing part of the Arabidopsis root. In this study, we provide a more accurate criterion to identify the MZ. Traditionally, the transition between the EZ to the MZ has been established by the emergence of the first root-hair bulge in the epidermis, because this emergence coincides with cell maturation in this cell type. But we have found here that after the emergence of the first root-hair bulge some cells continue to elongate and we have confirmed this in three different Arabidopsis ecotypes. We established the limit between the EZ and the MZ by looking for the closest cortical cell with a longer length than the average cell length of 10 cells after the cortical cell closest to the epidermal cell with the first root-hair bulge in these three ecotypes. In Col-0 and Ws this cell is four cells above the one with the root hair bulge and, in the Ler ecotype, this cell is five cells above. To unambiguously identifying the site at which cells stop elongating and attain their final length and fate at the MZ, we propose to calculate the length of completely elongated cortical cells counting 10 cells starting from the sixth cell above the cortical cell closest to the epidermal cell with the first root-hair bulge. We validated this proposal in the three ecotypes analyzed and consider that this proposal may aid at having a more objective way to characterize root phenotypes and compare among them.

A pot experiment was conducted on spring maize to determine the effect of the water deficit period and nitrogen application rate on leaf protective system of malondialdehyde (MDA), soluble sugar (SS) contents as well as peroxidase (POD) and superoxide dismutase (SOD) activity. The study shows that while regulated deficit irrigation increases MDA and SS contents, it at the same time decreases POD and SOD activity. Nitrogen application ensures high levels of SS, SOD and POD before heading. MDA content is negatively correlated with nitrogen rate and irrigation level, reaching its peak in the late stage of jointing, and then decreasing. Nitrogen application in regulated deficit irrigation leads to a relatively high SS content from seedling to terminal jointing stage, whereas high nitrogen application rates in continuous water deficit decreases SS content. POD activity is maximum at terminal jointing stage, decreasing during heading stage. Water deficit significantly decreases POD activity, with a significant compensation effect after re-flood irrigation. Although nitrogen application enhances POD activity, but high nitrogen dose with water deficit in heading or during whole growing stage decreases POD activity at heading stage. SOD activity trend is consistent with that of POD. Compensation effect of re-flood irrigation is better for physiology and biochemistry under water deficit at seedling stage, and it takes the second place under water deficit at jointing stage. High nitrogen treatment is not suitable for water deficit irrigation at heading stage. The best treatment combination is water deficit at seedling stage with low nitrogen dose.

The soil-borne Gram-negative β-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding β-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.

MAIN CONCLUSION: miRNAs control leaf size of maize crop during chilling stress tolerance by regulating developmentally important transcriptional factors and sustaining redox homeostasis of cells. Chilling temperature (0-15°C) is a major constraint for the cultivation of maize (Zea mays) which inhibits the early growth of maize leading to reduction in leaf size. Growth and development take place in meristem, elongation, and mature zones that are linearly located along the leaf base to tip. To prevent shortening of leaf caused by chilling, this study aims to elucidate the regulatory roles of microRNA (miRNA) genes in the controlling process switching between growth and developmental stages. In this respect, hybrid maize ADA313 seedlings were treated to the chilling temperature which caused 26% and 29% reduction in the final leaf length and a decline in cell production of the fourth leaf. The flow cytometry data integrated with the expression analysis of cell cycle genes indicated that the reason for the decline was a failure proceeding from G2/M rather than G1/S. Through an miRNome analysis of 321 known maize miRNAs, 24, 6, and 20 miRNAs were assigned to putative meristem, elongation, and mature zones, respectively according to their chilling response. To gain deeper insight into decreased cell production, in silico, target prediction analysis was performed for meristem specific miRNAs. Among the miRNAs, miR160, miR319, miR395, miR396, miR408, miR528, and miR1432 were selected for confirming the potential of negative regulation with their predicted targets by qRT-PCR. These findings indicated evidence for improvement of growth and yield under chilling stress of the maize.

Heavy metals are considered important environmental contaminants, and their mixture toxicity on plants has complex mutual interactions. The interactive effects of heavy metals on growth, photosynthetic parameters, lipid peroxidation and compatible osmolytes were studied in Kandelia obovata grown for 5months in sediment treated with combinations of lead (Pb), zinc (Zn) and copper (Cu). The results showed no significant reduction of biomass under heavy metal stresses, except for decreased root biomass under higher Pb + Cu treatment, indicating high tolerance of K. obovata to heavy metal stress. Only the photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), decreased with increasing concentration of treatments (except for Pb + Cu and Pb + Zn + Cu). Trinary treatment (Pb + Zn + Cu) increased biomass and the photosynthetic parameters when compared to the external addition of binary metals. In the roots, biomass and soluble sugar content were lower under binary than trinary treatments, indicating that the combination of Zn and Cu exhibited improved effects of alleviating toxicity than each of them alone in Pb-containing combined treatments. In the leaves, Zn-containing combined treatments significantly decreased malondialdehyde (MDA), soluble sugar and proline content in low concentration, while Pb + Cu treatments significantly increased these parameters (P < 0.05). The correlation analysis showed that leaf MDA and proline content were negatively correlated with Zn concentration (P < 0.05). Zn could alleviate the effects of combined heavy metal stress, and Pb + Cu treatment showed synergistic effects in leaves. The positive correlations between MDA content and the osmotic parameters showed that osmotic stress and lipid membranes oxidation exist simultaneously under multiple heavy metal stresses. Therefore, biomass, Tr, leaf MDA, leaf proline content and soluble sugar content could indicate metal mixture toxicity to mangrove seedlings.