Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.     

Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp

Four filamentous ascomycetes, Leptosphaeria maculans, L. biglobosa, Oculimacula yallundae and O. acuformis, were transformed via Agrobacterium tumefaciens-mediated transformation with the genes encoding DsRed and GFP. Using vectors pCAMDsRed and pCAMBgfp, either germinated conidia of Leptosphaeria spp. and O. yallundae or physically fragmented cultures of Oculimacula spp. were transformed. In vitro, the expression of the two reporter proteins in mycelium of both Oculimacula and both Leptosphaeria species was sufficient to distinguish each species in co-inoculated cultures. In planta, transformants of L. maculans or L. biglobosa expressing DsRed or GFP could be observed together in leaves of Brassica napus. Either reporter protein could be used to view the colonization of leaf petioles by both Leptosphaeria spp. and growth in the xylem vessels could be clearly observed. With the generation of these transformants, further studies on interactions between pathogen species involved in disease complexes on various host species and between opposite mating types of the same species are now possible.     

Recognition of avirulence gene AvrLm1 from hemibiotrophic ascomycete Leptosphaeria maculans triggers salicylic acid and ethylene signaling in Brassica napus

Interaction of a plant with a fungal pathogen is an encounter with hundreds of molecules. In contrast to this, a single molecule often decides between the disease and resistance. In the present article, we describe the defense responses triggered by AvrLm1, an avirulence gene from a hemibiotrophic ascomycete, Leptosphaeria maculans, responsible for an incompatible interaction with Brassica napus. Using multiple hormone quantification and expression analysis of defense-related genes, we investigated signaling events in Rlm1 plants infected with two sister isolates of L. maculans differentiated by the presence or absence of AvrLm1. Infection with the isolate carrying AvrLm1 increased the biosynthesis of salicylic acid (SA) and induced expression of the SA-associated genes ICS1, WRKY70, and PR-1, a feature characteristic of responses to biotrophic pathogens and resistance gene-mediated resistance. In addition to SA-signaling elements, we also observed the induction of ASC2a, HEL, and CHI genes associated with ethylene (ET) signaling. Pharmacological experiments confirmed the positive roles of SA and ET in mediating resistance to L. maculans. The unusual cooperation of SA and ET signaling might be a response to the hemibiotrophic nature of L. maculans. Our results also demonstrate the profound difference between the natural host B. napus and the model plant Arabidopsis in their response to L. maculans infection.     

MORPHOLOGY OF HUMULUS LUPULUS. II. SECONDARY GROWTH IN THE ROOT AND SEEDLING VASCULARIZATION

Miller , Robert H. (U. Nevada, Reno.) Morphology of Humulus luppulus. II. Secondary growth in the root and seedling vascularization. Amer. Jour. Bot. 46(4): 269–277. Illus. 1959.—In the primary state the roots of Humulus lupulus L. have a diarch xylem plate with 2 strands of primary phloem lying on either side of the primary xylem. Secondary histogenesis is described for the primary root. Fibrous and fleshy storage roots are developed by the hop plant and their respective developmental and anatomical structures are described. Lateral roots are initiated in the pericycle opposite the protoxylem poles. The architecture of these secondary roots is similar to that of the primary root. The seedling develops a fleshy storage organ through secondary growth of the primary root and the hypocotyl. The hypocotyl eventually resembles a fleshy taproot throughout most of its extent. The vascular cambium differentiates large amounts of parenchymatous tissues. A relatively smaller amount of tracheary tissue is formed. The secondary phloem comprises a high percentage of phloem parenchyma and ray cells containing numerous large starch grains, and constitutes the larger portion of the fleshy storage root. Numerous thick-walled lignified fibers occur throughout the secondary vascular tissues. Resin and tannin cells are abundantly distributed. A phellogen is differentiated from the pericycle and develops a persistent periderm on the outer surface of the fleshy storage organ. A relatively short transition region occurs in the upper part of the hypocotyl. The transition takes place from a radially alternate arrangement of the vascular tissues in the root to a collateral arrangement in the cotyledons.     

Spread of beet necrotic yellow vein virus in infected seedlings and plants of sugar beet (Beta vulgaris)

Summary Infection of sugar beet roots by beet necrotic yellow vein virus (BNYVV) was investigated with transmission electron microscopy, immunogold labelling and enzyme linked immuno sorbent assay (ELISA). Here we show that infection of sugar beet roots is very fast, occurring during germination. Seedlings grown directly in infected soil showed higher BNYVV infection than plants transplanted into infected soil after seven days of initial growth in sterilized soil. The earlier the initial infection, the faster was its spread. The study showed that a few differentiated cells of the cortex and of the xylem parenchyma were the preferred sites of viral multiplication. The spread of viral infection was slow through differentiated tissues. Intact virions were frequently found in undifferentiated and mature vessel elements and xylem parenchyma, whereas they were rare in sieve elements. Virus particle number in the differentiating tracheary elements was high, suggesting that infection of the vessel elements preceded their differentiation. This would explain increased infection after early inoculation. Even the xylem tissue of the primary root was highly infected, the seedlings lacked virus particles in their hypocotyls and leaves.     

Regeneration of plants from callus tissues of hairy roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi

INTRoDUCTIONThe hairy root disease is a patholOgical syndromeof dicotyledonous plants fOllowing wounding and in-fection with Agrobacterum rhjZOgenesI1]. The rhi-zogenicity is conferred to p1ant cel1s by a fragmentof DNA (Ri T-DNA), which is transferred from thelarge root--inducting (Ri) plasmid, haJrboured by thebacterium, to the genome, where it is stably inte-grated and expressed. Illtegration of a DNA segment(T-DNA) of pRi into the host genome 1eads to ac-tive proliferation of…     

A trans-zeatin riboside in root xylem sap negatively regulates adventitious root formation on cucumber hypocotyls

Shoot cultures of cucumber were used to analyse the roles of root-derived substances in adventitious root formation on hypocotyl tissues. Xylem sap collected from the roots of squash had a strong inhibitory effect on the formation of hypocotyl adventitious roots. Double-solvent extraction followed by fractionation with both normal and reverse phase column chromatographies and analysis by liquid chromatography/tandem mass spectrometry identified trans-zeatin riboside (ZR) as the primary suppressor of adventitious root formation. ZR was the predominant cytokinin present in the xylem sap, occurring at a concentration of 2x10(-8 )M. Application of ZR at concentrations from 3.16x10(-9) M effected inhibition of adventitious root formation. These results suggest that ZR transported from roots via xylem sap may act as an endogenous suppressor of hypocotyl adventitious root formation in planta.     

Infection, propagation, distribution and stability of plant virus in hairy root cultures

Nicotiana benthamiana hairy root cultures were infected with tobacco mosaic virus (TMV) and used for in vitro plant virus propagation. The roots were infected with TMV by addition of virus to the medium at the same time as root inoculation. Viral accumulation in the biomass was 7-11-fold greater when the initial infection was carried out in B5 medium rather than sodium phosphate buffer; virus accumulation also increased with increasing viral inoculum concentration. The amount of TMV accumulated in the biomass was similar when virus was retained in the medium for the duration of the cultures and when the inoculum virus was removed 23h after addition to the roots. In roots with established infections, the concentration of virus remained relatively constant and did not increase with further root growth. The distribution of virus within individual root mats harvested from shake flasks was not uniform; there was also significant variability in viral accumulation between replicate hairy root cultures. The picture that emerges from this work is that in vitro viral accumulation in hairy root cultures depends strongly on the viral inoculum concentration applied and the initial level of primary infection achieved, even though primary infection by external virus occurs mainly within only the first few hours of exposure to the biomass and is followed by substantial secondary infection by viral progeny within the root tissue.     

类短命植物阿尔泰独尾草的解剖学研究

通过常规石蜡切片法,对类短命植物阿尔泰独尾草的根、茎、叶、花等器官进行了解剖结构的研究。结果表明：阿尔泰独尾草的根系有明显的二型性,即有储藏根和吸收根的结构与功能分化,是其对生长发育快速、年休眠期长的类短命植物生活习性高度适应的结果;其营养器官表现出明显的旱生植物特征;储藏根与吸收根木质部的二型性及其内皮层的带状凯氏带增厚结构则说明独尾草属植物可能具有较为特殊的系统演化地位。     

Leptosphaeria maculans elicits apoptosis coincident with leaf lesion formation and hyphal advance in Brassica napus

Programmed cell death, with many of the morphological markers of apoptosis, is increasingly recognized as an important process in plant disease. We have investigated the involvement and potential role of apoptosis during the formation of leaf lesions by the fungus Leptosphaeria maculans on susceptible Brassica napus cv. Westar. There were no signs of host cell damage until 7 to 8 days postinoculation (dpi), when trypan-blue-stained leaf mesophyll cells were first detected. Hyphae were visible in the intercellular spaces of the inoculated area from 5 dpi and were associated with trypan-blue-stained cells at 8 to 9 dpi. Hallmarks of apoptosis, observed coincident with or immediately prior to the formation of leaf lesions at 8 to 10 dpi, included membrane shrinkage of the mesophyll cell cytoplasm, loss of cell to cell contact in mesophyll cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of nuclei in apparently "healthy" tissue immediately adjacent to dead areas. Hyphae were highly branched and prolific in the "healthy" tissue immediately adjacent to dead areas 9 to 10 dpi, and formed pycnidia inside dead areas 11 to 12 dpi. Coinfiltration of the tetrapeptide caspase inhibitor Ac-DEVD-CHO with spores of the pathogen significantly suppressed development of leaf lesions but did not affect fungus viability. We hypothesize that L. maculans elicits apoptosis as a dependent component of pathogenesis in susceptible B. napus, and that the fungus uses apoptotic cells as a source of nutrition for reproduction and further growth.     

Flowering as a condition for xylem expansion in Arabidopsis hypocotyl and root

In dicotyledons, biomass predominantly represents cell-wall material of xylem, which is formed during the genetically poorly characterized secondary growth of the vasculature. In Arabidopsis hypocotyls, initially proportional secondary growth of all tissues is followed by a phase of xylem expansion and fiber differentiation. The factors that control this transition are unknown. We observed natural variation in Arabidopsis hypocotyl secondary growth and its coordination with root secondary growth. Quantitative trait loci (QTL) analyses of a recombinant inbred line (RIL) population demonstrated separate genetic control of developmentally synchronized secondary-growth parameters. However, major QTL for xylem expansion and fiber differentiation correlated tightly and coincided with major flowering time QTL. Correlation between xylem expansion and flowering was confirmed in another RIL population and also found across Arabidopsis accessions. Gene-expression analyses suggest that xylem expansion is initiated after flowering induction but before inflorescence emergence. Consistent with this idea, transient activation of an inducer of flowering at the rosette stage promoted xylem expansion. Although the shoot was needed to trigger xylem expansion and can control it in a graft-transmissible fashion, the inflorescence stem was not required to sustain it. Collectively, our results suggest that flowering induction is the condition for xylem expansion in hypocotyl and root secondary growth.     

Ascospore Discharge, Leaf Infestation and Variations in Pathogenicity as Criteria to Predict Impact of Leptosphaeria maculans on Oilseed Rape

The aim of this investigation was to evaluate criteria for the forecast and targeted control of basal stem canker (blackleg) caused by Leptosphaeria maculans on oilseed rape. Ascospore discharge, ratio of aggressive and non-aggressive isolates and leaf and stem infestations were determined during 1991/92–1993/94 at 6–10 sites in Northern Germany. On a 1–9 scale, blackleg intensity varied from 2.3 to 6.3 at BBCH 81 between different sites and years. Ascospore discharge started in September or October, and reached maxima 1 or 2 months later, without an apparent relationship to blackleg or leaf infestation. There was a positive relationship between leaf infestation and blackleg. However, correlation coefficients were too low to be used as a basis for forecasting. On plant residues from the stem base, aggressive isolates were dominant (>80%) on all sites. From higher parts of the stem and from leaves also, non-aggressive isolates were isolated with higher frequencies on some locations, but the proportion of aggressive isolates was not related to the blackleg intensity. Taken all together, the three criteria alone seem to be insufficient for the development of a system of blackleg forecasting and targeted control. Further factors (e.g. climatic factors, seed-and soilborne inoculum, cultural practices) have to be included in models for forecasting the impact of blackleg on oilseed rape.     

Probing crucial metabolic pathways in fungal pathogens of crucifers: biotransformation of indole-3-acetaldoxime, 4-hydroxyphenylacetaldoxime,and their metabolites

Indole-3-acetaldoxime is an intermediate of crucial importance in the biosynthesis of diverse plant secondary metabolites of Cruciferae. The metabolism of indole-3-acetaldoxime to indole-3-acetic acid via indole-3-acetonitrile by fungi that cause important plant diseases in crucifers, Leptosphaeria maculans (asexual stage Phoma lingam) causative agent of blackleg disease, Rhizoctonia solani causative agent of root rot disease, and Sclerotinia sclerotiorum causative agent of stem rot disease, is described. As well, the antifungal activity of indole-3-acetaldoxime and metabolites and the synthesis and biotransformation of 4-hydroxyphenylacetaldoxime by the same plant pathogens and by an insect fungal pathogen, Beauveria bassiana, are reported.     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

Anatomical Studies on Hypocotyl of Circaeaster

The stem of Circaeaster agrestis Maxim. is very short but the length of hypocotyl is comparatively long, almost occupying the whole length of the plant. This tender hypocotyl is mainly supported by the thickening of cuticle on the outer wall of the epidermal cell and the primary xylem in the center. Between primary xylem and primary phloem there are 2–3 layers of parenchymatous cells, regularly or irregularly arranged, but no cambial zone can be recognized. The transition region where root and stem meet showed no evidence of twisting, splitting or inversion of the strands in the primary vascular tissues which are common in most of the dicots. The extending cotyledon traces differentiate directly from the parenchymatous cells which locate on the outside of the poles of primary xylem. The first and the second leaf traces are organized in the middle of the primary phloem.     

Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change

Leptosphaeria maculans is the ascomycete responsible for one of the most damaging diseases of oilseed rape ( Brassica napus ), stem canker of crucifers. Both avirulence ( AvrLm ) genes in the fungus and resistance ( Rlm ) genes in the plant are genetically clustered. Using a map-based cloning strategy, we delineated a 238 kb region containing the AvrLm7 locus. Structural features of the region were reminiscent of those previously found on another chromosome for genomic regions encompassing AvrLm1 and AvrLm6 , i.e. GC-equilibrated, gene-rich isochores alternating with AT-rich, recombination-deficient, gene-poor isochores. These latter corresponded to mosaics of degenerated and truncated transposable elements. AvrLm7 is the only gene located within a 60 kb AT-rich isochore. It induced resistance responses in plants harbouring either Rlm7 or Rlm4 , and was thus renamed AvrLm4-7 . It encodes a 143-amino-acid cysteine-rich protein, predicted to be secreted, and strongly induced during early stages of plant infection. Sequencing and restriction analyses of AvrLm4–AvrLm7 or avrLm4–AvrLm7 alleles in L. maculans field isolates, and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine to an arginine residue, was responsible for the loss of AvrLm4 specificity whereas AvrLm7 recognition was unaltered.