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The main restraint obstructing the wider adoption of lupins as protein crops is the presence of bitter and toxic quinolizidine alkaloids (QAs), whose contents might increase under exposure to stressful environmental conditions. A poor understanding of how QAs accumulate hinders the breeding of sweet varieties. Here, we characterize the expression profiles of QA-related genes, along with the alkaloid content, in various organs of sweet and bitter narrow-leafed lupin (NLL, Lupinus angustifolius L.). Special attention is paid to the RAP2-7 transcription factor, a candidate regulator of the QA pathway. We demonstrate the upregulation of RAP2-7 and other QA-related genes, across the aerial organs of a bitter cultivar and the significant correlations between their expression levels, thus supporting the role of RAP2-7 as an important regulatory gene in NLL. Moreover, we showed that the initial steps of QA synthesis might occur independently in all aerial plant organs sharing common regulatory mechanisms. Nonetheless, other regulatory steps might be involved in RAP2-7 -triggered QA accumulation, given its expression pattern in leaves. Finally, the examination of QA-related gene expression in plants infected with Colletotrichum lupini evidenced no connection between QA synthesis and anthracnose resistance, in contrast to the important role of polyamines during plant-pathogen interactions.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Glehnia littoralis is an endangered medicinal plant growing in the coastal ecological environment and plays an important role in coastal ecosystems. The endophytes in the plant have a significant role in promoting plant growth and enhancing plant stress resistance. However, the endophytic bacterial structure associated with halophyte G. littoralis is still not revealed. In this project, the construction and diversity of endophytic bacterial consortium associated with different tissues of G. littoralis were illustrated with high throughput sequencing of the V3-V4 region of the bacterial 16S rRNA. The results resolved that the diversity and richness of endophytic bacteria were significantly higher in root than in leaf and stem. The operational taxonomic units (OTU) analysis demonstrated that the Actinobacteria and Proteobacteria were dominant in all the samples at the phylum level, and Pseudomonas, Bacillus, Rhizobium were the dominant genera. Our results unraveled that the bacterial communities differed among different tissues of G. littoralis . Endophytic bacterial communities in leaf and stem shared more similarity than that in the root. Furthermore, the difference of bacteria community and structure among different tissues were also detected by principal coordinate analysis. Taken altogether, we can conclude that the bacterial communities of different tissues are unique, which could facilitate understanding the diversity of endophytic bacteria in G. littoralis ., Competing Interests: Conflict of interest The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication., (© 2020 Xiaowei Huo et al.)

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. 'Hondia') and Triticum spelta L. (cv. 'Rokosz'), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1). It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the 'Hondia' cv., whereas Pedobacter and Duganella constituted the core microbiome in the 'Rokosz' cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Highlights: Small molecules (SMs) 1, 3, 4, and 5 are novel growth inhibitors of Salmonella enterica . These SMs are not toxic to tomato plant tissues including fruits. Combining biocontrol agents and SMs enhanced the control of Salmonella in infected plants. These SMs may be safe bactericides against Salmonella and phytopathogens in produce.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8., Competing Interests: The authors have declared that no competing interests exist.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Tea white scab (TWS) is a major disease affecting tea trees in mid-elevation regions and often occurs during rainy seasons with low temperatures. This disease is caused by the fungal pathogen Phoma sp. TWS can infect young stems, tender leaves, and tender shoots and lead to the production of low-quality tea. Owing to the absence of an effective control, TWS can result in substantial loss in tea production. In this study, we isolated and identified the pathogen from tea leaves infected by TWS and then evaluated in vitro the antifungal activity of Shenqinmycin, polyoxin, azoxystrobin, oligosaccharins, and tebuconazole against Phoma sp. Our results indicated that Shenqinmycin can inhibit the growth of Phoma sp. mycelia, with the EC 50 value of 0.74μg/mL. After Phoma sp. being incubated in PDB liquid medium with Shenqinmycin, its mycelia were distorted and distended at 1.56μg/mL of minimum inhibitory concentration for 6h. Crucial genes associated with cell redox homeostasis, proteins synthesis, energy metabolism, and cytoskeleton were studied at mRNA and protein levels through RT-qPCR and Nano-LC-MS/MS. The results showed that the genes of 3-phosphate-glyceraldehyde dehydrogenase, citrate synthase, NADH-ubiquinone oxidoreductase subunit (NADH-subunit), ribosomal protein, eukaryotic initiation factor 4A-I, β-tubulin, and α-tubulin were up-regulated. Meanwhile, the genes of formate dehydrogenase (FDH), malate dehydrogenase, mitochondrial heat shock protein, and protein disulfide-isomerase (PDI) were up-regulated at mRNA level but down-regulated at protein level. These results indicated that Shenqinmycin contribute to cell redox homeostasis by up- or down-regulating NADH-subunit, FDH, and PDI., (Copyright © 2017. Published by Elsevier Inc.)

This study describes 32 fungal endophytes isolated from different tissues of Brugmansia aurea Lagerh. Each fungal strain was authenticated based on internal transcribed spacer rDNA sequence. Phylogenetic analysis showed that these fungi are distributed in three classes, seven orders and 12 genera. The dichloromethane extracts of endophytic strains were screened for anticancer and antimicrobial activity. Anticancer activity of extracts against human cancer cell lines revealed that 50% strains are active with IC 50  < 10 μg/mL. While analysing antimicrobial potential against both Gram-positive and Gram-negative bacteria, 56.25% endophytic strains displayed activity at least against one of the tested human pathogenic bacteria with minimum inhibitory concentration of 12.5-100 μg/mL. In vitro antagonistic activity of endophytes was analysed against Sclerotinia sp ., Aspergillus fumigatus, Fusarium solani, A. flavus and F. oxysporum pathogen . The broad-spectrum anti-phytopathogenic activity was shown by R2BA. The presence of ketoacyl synthase domain of polyketide synthase gene and high degree of bioactivity shown by endophytic fungi suggested that they have potential to produce therapeutic compounds and to serve as biocontrol agent., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The genome and transcriptome sequences of the aquatic, rootless, and carnivorous plant Utricularia gibba L. (Lentibulariaceae), were recently determined. Traps are necessary for U. gibba because they help the plant to survive in nutrient-deprived environments. The U. gibba's traps (Ugt) are specialized structures that have been proposed to selectively filter microbial inhabitants. To determine whether the traps indeed have a microbiome that differs, in composition or abundance, from the microbiome in the surrounding environment, we used whole-genome shotgun (WGS) metagenomics to describe both the taxonomic and functional diversity of the Ugt microbiome. We collected U. gibba plants from their natural habitat and directly sequenced the metagenome of the Ugt microbiome and its surrounding water. The total predicted number of species in the Ugt was more than 1,100. Using pan-genome fragment recruitment analysis, we were able to identify to the species level of some key Ugt players, such as Pseudomonas monteilii. Functional analysis of the Ugt metagenome suggests that the trap microbiome plays an important role in nutrient scavenging and assimilation while complementing the hydrolytic functions of the plant.

The present study reports on the biotransformation of the brewer's spent grain (BSG) in co-digestion with Jerusalem artichoke (JA, Helianthus tuberosus L.) phytomass by thermophilic (+55 °C) and mesophilic (+30 °C) anaerobic methanogenic communities. BSG is a by-product of the beer-brewing process generated in large amounts, in which utilization provokes a negative effect on the environment. In this study, we will show an effective conversion of BSG into biogas by selected microbial communities, obtained from different sources (animal manure and previously isolated microbial consortia). The stimulation of methanogenesis was reached by the co-digestion of JA's phytomass (stem and leaves). The optimized conditions for microbial stable cultivation included the use of nutrient medium, containing yeast extract and trace element solution. The optimal BSG concentration in biogas production was 50 and 100 g L(-1). Under thermophilic conditions, the maximum total methane production reached 64%, and it comprised around 6-8 and 9-11 of L CH4 per 100 g of fermented BSG without and with co-digested JA, respectively, when the fresh inoculum was added. Although, after a year of re-cultivation, the values reduced to around 6-7, and 6-10 L CH4/100 g BSG, correspondingly, the selected microbial communities showed effective biotransformation of BSG. The supplementation of soil with the residual fermented BSG (10%, w/w) resulted in the promotion of lettuce (Lepidium sativum L.) growth. The results obtained demonstrate a potential for complete BSG utilization via biogas production and application as a soil additive.

Recent findings have shown that antibiotic resistance is widespread in multiple environments and multicellular organisms, as plants, harboring rich and complex bacterial communities, could be hot spot for emergence of antibiotic resistances as a response to bioactive molecules production by members of the same community. Here, we investigated a panel of 137 bacterial isolates present in different organs of the medicinal plant Echinacea purpurea, aiming to evaluate if different plant organs harbor strains with different antibiotic resistance profiles, implying then the presence of different biological interactions in the communities inhabiting different plant organs. Data obtained showed a large antibiotic resistance variability among strains, which was strongly related to the different plant organs (26% of total variance, P < 0.0001). Interestingly this uneven antibiotic resistance pattern was present also when a single genus (Pseudomonas), ubiquitous in all organs, was analyzed and no correlation of antibiotic resistance pattern with genomic relatedness among strains was found. In conclusion, we speculate that antibiotic resistance patterns are tightly linked to the type of plant organ under investigation, suggesting the presence of differential forms of biological interaction in stem/leaves, roots and rhizosphere., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.

The endoperoxide sesquiterpene lactone artemisinin which is isolated from the plant Artemisia annua, and its semi-synthetic derivatives, are potent, novel, antimalarial drugs. They are effective against multidrug-resistant Plasmodium strains and have become essential components of the so-called Artemisinin-based Combination Therapy, that is recommended by the World Health Organization as the treatment of choice for malaria tropica. Moreover, artemisinin and its derivatives show additional anti-parasite, antitumor, and anti-viral properties. The plants, however, are very poor resources for the drug, as the content of artemisinin is low (from 0,1 to 1,5 % of dried leaves) and dependent on seasonal and somatic variations as well as the infestation of bacteria, fungi and insects. A chemical synthesis of the compound is complex and uneconomic. Therefore, artemisinin is in short supply and remains unaffordable for most people in malaria-endemic countries. Thus, many researchers have focused on enhancing the production of artemisinin, first, through traditional breeding and in in vitro plant tissue cultures and, then, by heterologous expression systems (a semi-synthetic approach) with the use of genetically-modified or transgenic microbes. In this review, we summarize the progress made in the production of artemisinin by the biotechnological approach.

Background: Conservation tillage promotes the accretion of soil organic matter and often leads to improved soil fertility and moisture availability. However, few studies have looked at the physiological response of crop plants to different tillage practices. It was therefore hypothesised that measuring the nutrient concentrations and stable isotope composition (δ(13)C, δ(18)O, δ(15)N) of shoots could help evaluate the physiological response of common bean (Phaseolus vulgaris L.) to different tillage treatments (no tillage (NT) and mouldboard ploughing (MP)) in a rain-fed farming system in northern Mexico., Results: NT significantly enhanced shoot phosphorus concentration in bean plants. Tillage exerted a negative effect on the extent of root colonisation (%) by arbuscular mycorrhizal fungi (AMF). Lower shoot δ(18)O but unchanged δ(13)C values in plants from the NT system suggest enhanced stomatal conductance but also enhanced photosynthetic rate, which overall resulted in unchanged water use efficiency. Bean plants in the NT system showed lower shoot δ(15)N values, which suggests that a larger proportion of total plant nitrogen was obtained through atmospheric nitrogen fixation in this treatment., Conclusion: Greater diversity of AMF soil communities and heavier colonisation of roots by AMF in the NT compared with the MP system appeared to contribute to improved crop nutrition, water relations and yield in this rain-fed agroecosystem., (2010 Society of Chemical Industry.)

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

AFM is being applied in increasingly wide research fields and extracting more biochemical/biophysical information that is beyond the capability of traditional SEM and TEM. Due to its inherent features, AFM is rarely used to observe the subcellular details within cells. Although subcellular features were recently observed on thin sections of plant tissues using AFM, this method might introduce unexpected artifacts during sample processing. Here we try to observe plant cells still embedded in resin block. This modified method minimizes the possibility of artifacts. The comparison among outcomes of AFM, SEM, TEM and LM on the same single cell suggest that this modified method is a good, applicable, efficient and faithful way applying AFM on biological materials.

Traditional Chinese medicinal plants (TCMPs), commonly used as spices, additives or foods, are also widely used in China to prevent and cure human disease. Due to their provenance, TCMPs may be contaminated by various fungal species, including ochratoxigenic fungi, during growth, collection, transportation and, especially, storage. A reliable method for the determination of ochratoxin A (OTA) in TCMPs of different origins was developed to monitor OTA levels in China. Analyses involved the extraction of OTA by methanol/water, immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD). Positive results were further confirmed by LC-ESI-MS/MS. The limit of detection (LOD) was 0.3 microg kg(-1), based on a signal-to-noise ratio of 3 : 1. Among the total of 57 TCMPs samples collected from six different areas, 31 were visibly moldy due to inappropriate storage; 26 sample, purchased from local TCMPs markets, were not visibly moldy. The results showed that 23 of the visibly moldy samples and two of the not visibly moldy were contaminated with OTA at levels ranging 1.2-158.7 and 2.5-5.6 microg kg(-1), respectively. This is the first report of the natural occurrence of OTA in TCMPs.

The cultivation of genetically modified Bt maize has raised environmental concerns, as large amounts of plant residues remain in the field and may negatively impact the soil ecosystem. In a field experiment, decomposition of leaf residues from three genetically modified (two expressing the Cry1Ab, one the Cry3Bb1 protein) and six non-transgenic hybrids (the three corresponding non-transformed near-isolines and three conventional hybrids) was investigated using litterbags. To elucidate the mechanisms that cause differences in plant decomposition, structural plant components (i.e., C:N ratio, lignin, cellulose, hemicellulose) were examined. Furthermore, Cry1Ab and Cry3Bb1 protein concentrations in maize leaf residues were measured from harvest to the next growing season. While leaf residue decomposition in transgenic and non-transgenic plants was similar, differences among conventional cultivars were evident. Similarly, plant components among conventional hybrids differed more than between transgenic and non-transgenic hybrids. Moreover, differences in senescent plant material collected directly from plants were larger than after exposure to soil for 5 months. While the concentration of Cry3Bb1 was higher in senescent maize leaves than that of Cry1Ab, degradation was faster, indicating that Cry3Bb1 has a shorter persistence in plant residues. As decomposition patterns of Bt-transgenic maize were shown to be well within the range of common conventional hybrids, there is no indication of ecologically relevant, adverse effects on the activity of the decomposer community.

Endophytic fungi are rich in species diversity and may play an important role in the fitness of their host plants. This study investigated the diversity and antimicrobial potential of endophytic fungi obtained from Saussurea involucrata KAR. et KIR. A total of 49 endophytic fungi were isolated from S. involucrata and identified using morphological and molecular techniques. Extracts of fermentation broth from the 49 fungi were tested for antimicrobial activity against pathogenic microorganisms using the agar diffusion method. Forty-eight out of the 49 endophytic fungi were identified and grouped into 14 taxa. Cylindrocarpon sp. was the dominant species isolated from S. involucrata, followed by Phoma sp. and Fusarium sp. Among the 49 endophytic fungi, 9 root isolates having darkly pigmented, septate hyphae were identified as dark septate endophytic (DSE) fungus, and 12 fungi inhibited at least one test microorganism. Moreover, 5 strains showed a broader spectrum of antimicrobial activity and 4 strains displayed strong inhibition (+++) against pathogenic fungi. The results indicate that endophytic fungi isolated from S. involucrata are diverse in species and a potential source of antimicrobial agents.

A comparative analysis of cellulase activity of 36 fungal strains of phytopathogenic and endophytic Ceratocystis sp. was conducted. The rate of their linear growth on the media with carboxymethylcellulose was studied. It was shown that the rate of linear growth of phytopathogenic strains on the media with carboxymethylcellulose was lower than that in endophytic ones. There was no correlation between the levels of cellulase activity of studied strains and rates of their linear growth. The majority of Ceratocystis sp. strains had middle but not high cellulase activity, cellulase activity varied in the group of endophytic strains more than in the phytopathogenic one. The differences in cellulase activity were observed on the strain level. No distinct dependence of cellulase activity on different growth terms of fungi was demonstrated. The distinct dependence of cellulase activity level of endophytes on the species and organs of host plant was not observed. The cellulase activity level of endophytic strains Ceratocystis sp. was lower than in endophytic Fusarium poae strains and similar to it in phytopathogenic Fusarium strains. Consequently the investigated endophytic Ceratocystis sp. strains can be classified as latent pathogens, which were able to cause the diseases of host plants in favorable for them conditions. The paper is presented in Ukrainian.

Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine. Swainsonine accumulates in all parts of the plant with the highest concentrations found in the above ground parts. A fungal endophyte, Undifilum oxytropis, found in locoweed plant species, is responsible for the synthesis of swainsonine. By using quantitative PCR, the endophyte can be quantified in locoweed species. Endophyte amounts differ between plant parts and in some instances do not mirror the concentrations of swainsonine in the corresponding parts. Two groups of Oxytropis sericea were identified: one that accumulated high concentrations of swainsonine and another where swainsonine was not detected, or concentrations were near the detection threshold. The plants with high swainsonine concentrations had quantitatively higher amounts of endophyte. Alternatively, plants with low or no swainsonine detected had quantitatively lower endophyte amounts. In addition, swainsonine and endophyte concentrations were not distributed uniformly within the same plant when separated into stalks (leaves, scape(s), and flowers/pods). These findings provide evidence as to why plants in the same population accumulate different concentrations of swainsonine, and they have important implications for sampling of locoweed plants.

Different organs of 155 samples of conditionally healthy and sick oaks were examined for mycological contamination. The isolated microscopic fungi belonged to 129 species of different taxonomic groups. The pathogenic complex was represented by 30 fungi species, however most of these species were isolated in single cases. Roots, wood, bast and bark were not affected by the phytopathogenic fungi species that can cause the death of the trees. Mycobiota of branches and sprouts was similar to that ofbark and wood. But frequency of occurence of phytopathogenic fungi was higher on the branches and leaves of drying out and dried oaks. Phytopathogenic fungi Alternaria alternata, Ceratocystis sp. and Trichothecium roseum were isolated more often than others. More often occurrence of Ceratocystis sp.was observed during last two years, they could not be the basic cause of the oak drying. That is also confirmed by similarity of mycobiota of conditionally healthy and drying oaks.

In plants, various chitinases have been identified and categorized into several groups based on the analysis of their sequences and domains. We have isolated SafchiA, a novel class of chitinase from saffron (Crocus sativus L.). The cDNA encoding SafchiA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi Fusarium oxysporum, Beauveria and Phoma sp., suggesting a defence role of the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic, and antifungal activity. The deduced protein shares high similarity with chitinases belonging to family 19 of glycosyl-hydrolases, although some changes in the enzyme active site are present. To explore the properties of SafchiA we have expressed recombinant SafchiA in Escherichia coli and generated four different mutants affected in residues involved in the catalytic activity. One glutamic acid essential for family 19 chitinases activity is not present in C. sativus chitinase suggesting that only one acidic residue is necessary for the enzyme activity, in a similar manner as family 18 glycosyl-hydrolases.

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.

The occurrence of endophytic fungi in fennel, lettuce, chicory, and celery crops was investigated in southern Italy. A total of 186 symptomless plants was randomly collected and sampled at the stage of commercial ripeness. Fungal species of Acremonium, Alternaria, Fusarium, and Plectosporium were detected in all four crops; Plectosporium tabacinum was the most common in all crop species and surveyed sites. The effect of eight endophytic isolates (five belonging to Plectosporium tabacinum and three to three species of Acremonium) inoculated on lettuce plants grown in gnotobiosis was assessed by recording plant height, root length and dry weight, collar diameter, root necrosis, and leaf yellowing. P. tabacinum and three species of Acremonium, inoculated on gnotobiotically grown lettuce plants, showed pathogenic activity that varied with the fungal isolate. Lettuce plants inoculated with the isolates Ak of Acremonium kiliense, Ac of Acremonium cucurbitacearum, and P35 of P. tabacinum showed an increased root growth, compared to the non-inoculated control. The high frequency of P. tabacinum isolation recorded in lettuce plants collected in Bari and Metaponto, and in fennel plants from Foggia agricultural districts, suggests a relationship not only between a crop species and P. tabacinum, but also between the occurrence of the endophyte and the crop rotation history of the soil.

A severe canker disease has been causing decline and death of Corymbia calophylla in the southwest of Western Australia (WA) for some years, but the causal agent has never been investigated. However, there have been historical reports dating back to the 1920s of a canker disease of amenity planted C. ficifolia caused by 'Sporotrichum destructor', though the description and Latin diagnosis were never published. It has been suggested that there may be links between this species and the genus Quambalaria, a group containing leaf and shoot pathogens of species of Eucalyptus and Corymbia. The aim of this study was to investigate the identity of the pathogen historically attributed to canker disease of C. ficifolia, determine whether this pathogen is responsible for the current epidemic of C. calophylla canker, and whether it is synonymous with Quambalaria. Surveys examined the range of Quambalaria spp. on Corymbia spp. endemic to southwest WA. Their phylogenetic relationship to Q. cyanescens, Q. eucalypti, and Q. pitereka was examined using rLSU and ITS sequence data. Morphological characters were also compared. Sequences confirmed that Q. cyanescens and Q. pitereka are present in southwest WA, with the latter associated with leaf and shoot disease. A third group isolated from cankers represent a new species of Quambalaria. Comparisons of disease symptoms and conidiogenesis indicate this species is synonymous with 'S. destructor'. The species is formally described here as Q. coyrecup sp. nov.

This study was conducted to explore systemic infection by the Aspergillus flavus group into corn ears via the stalk. An A. parasiticus mutant which produces norsolorinic (NOR) acid (a visible orange intermediate of the aflatoxin biosynthetic pathway) was used in field studies to monitor systemic infection of corn stalk and ear tissues. Corn hybrids resistant and susceptible to aflatoxin contamination were grown in the field and inoculated prior to tasseling by inserting A. parasiticus infested toothpicks into stalks between the 5th and 6th node below the lowest ear shoot. Beginning 2 weeks after inoculation, systemic infection by the NOR mutant was assessed weekly by collecting ear shank tissue and stalk tissue from the nodes between the infection sites and the developing ears. Ears were collected at the end of the growing season to determine the level of kernel infection by the NOR mutant. In two separate studies, the A. parasiticus NOR mutant was isolated from stalk tissues at all of node positions and ear shank tissue from several susceptible corn hybrid plants at the first harvest date 2 weeks after inoculation. The NOR mutant was also isolated from stalk and ear tissue of a resistant hybrid. The NOR mutant was only isolated from kernels of susceptible hybrids in 2003 and 2004. Infection rates of kernels in infected ears were very low (<1%). In 2005, the fungus was found in only one kernel from an ear of the resistant hybrid. The NOR mutant was not isolated from stalks, ears, or kernels from control (uninoculated) plants grown in the plots with inoculated plants. Although infection levels of corn kernels were low, systemic movement of the A. parasiticus up the stalk appears to be another possible route to infection of developing corn ears.

A new full-length beta-1,3-glucanase cDNA, MpGlu, was isolated from a plantain (Musa paradisica) by the rapid amplification of cDNA ends (RACE) technique. Recombinant GST-MpGlu protein, expressed in E. coli, hydrolyzed (1-->3),(1-->6)-beta-glucan of Laminaria digitata and inhibited the growth of Fusarium oxysporum fsp. cubense (race 4) suggesting that it is a beta-1,3-glucanase. Southern blot analysis indicated that there is one copy of MpGlu in the plantain genome. MpGlu gene expression was detected in plantain leaves, peel, and pulp by RT-PCR. Northern blot analysis revealed that the expression of MpGlu was up-regulated by Fusarium infection. Subcellular localization analysis indicated that 28 residues at the N-terminal end are necessary for extracellular secretion, while 32 residues at the C-terminal end are necessary to target the protein into vacuoles.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10(4)-10(6) individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.

Tissues from Coffea arabica, C. congensis, C. dewevrei and C. liberica collected in Colombia, Hawaii and at a local plant nursery in Maryland were sampled for the presence of fungal endophytes. Surface sterilized tissues including roots, leaves, stems and various berry parts were plated on yeast-malt agar. DNA was extracted from a set of isolates visually recognized as Penicillium, and the internal transcribed spacer region and partial LSU-rDNA was amplified and sequenced. Comparison of DNA sequences with GenBank and unpublished sequences revealed the presence of 11 known Penicillium species: P. brevicompactum, P. brocae, P. cecidicola, P. citrinum, P. coffeae, P. crustosum, P. janthinellum, P. olsonii, P. oxalicum, P. sclerotiorum and P. steckii as well as two possibly undescribed species near P. diversum and P. roseopurpureum. Ochratoxin A was produced by only four isolates, one isolate each of P. brevicompactum, P. crustosum, P. olsonii and P. oxalicum. The role these endophytes play in the biology of the coffee plant remains enigmatic.

Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.

The influence of autoclaved fungal materials such as culture filtrate, freeze-dried mycelium (FDM), mycelium suspension, and spore suspension (SS) on the growth, morphogenesis, and carvone production of spearmint (Mentha spicata L.) plants was studied. Fungal materials were either applied as a drench or spray on the plants. Spearmint plants (cv. "294099") drenched with SS (1 x 10(8) spores/ml) of Trichoderma reesei showed no significant differences in leaf numbers, root numbers, or shoot numbers compared with nontreated controls. However, significantly higher fresh weights and carvone levels were observed in plants drenched with T. reesei SS compared with the untreated controls. Fungal materials derived from Aspergillus sp., Fusarium graminearum, F. sporotrichoides, Penicillium sp., P. acculeatum, Rhizopus oryzae, and T. reesei were sprayed on spearmint foliage. F. graminearum, F. sporotrichoides, or R. oryzae elicited no enhanced growth, morphogenesis, or secondary metabolism responses. The best growth and morphogenesis responses were obtained employing Aspergillus sp., Penicillium sp., or T. reesei foliar sprays. For example, spearmint cv. "557807" plants sprayed with 100 mg/l FDM T. reesei isolate NRRL 11460 C30 stimulated higher fresh weights (75%), shoot numbers (39%), leaf numbers (57%), and root numbers (108%) compared with untreated plants. This effect was not dose-dependent because similar growth and morphogenesis responses were obtained by testing 10, 100, or 1000 mg/l FDM concentrations. Carvone levels in fungal-treated foliar-sprayed plants were comparable to nontreated controls. However, total carvone levels per plant were higher in fungal-treated plants because of their increased fresh weight.

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.

The endophytic behaviour of Sclerotinia pseudotuberosa, cause of black rot of fruits of Castanea sativa, was investigated in asymptomatic tissues of the host, including nuts, buds and bark. A nested polymerase chain reaction (PCR) assay was developed for the specific detection of S. pseudotuberosa. Two specific primers, RAC1 and RAC2 were designed within the ITS1-5.8S-ITS2 region of rDNA by comparison of sequences of 24 isolates of S. pseudotuberosa and related species. Specificity of primers was tested against isolates of related and unrelated genera and the total DNA of C. sativa. The assay proved to be highly sensitive and capable of detecting picograms of S. pseudotuberosa DNA. Our study showed that S. pseudotuberosa was present asymptomatically in all the chestnut tissues tested. It is discussed that latency of S. pseudotuberosa in chestnut tissues may represent a adaptive strategy of the pathogen for rapid and massive host colonization in favourable environmental conditions.

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown., ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Plant-associated microbial diversity encompasses symbionts, protecting their host against various aggressions. Mycorrhizal and rhizospheric microorganisms buffer effects of soil toxic compounds and soil-borne pathogens. Endophytic bacteria and fungi, some of which are vertically inherited through seeds, take part in plant protection by acting directly on aggressive factors (mainly pathogens and herbivores) or by enhancing plant responses. Plant protective microbial symbionts determine the ecological success of plants; they drastically modify plant communities and related trophic webs. This review suggests approaches to improve the inventory of diversity and functions of in situ plant-associated microorganisms.

Yeast extract and Venturia inaequalis treated intact scab-susceptible (McIntosh) and scab-resistant (Liberty) apple plants and their organs were analyzed for phenolic metabolites. The major phenolic compounds found in both non-treated and treated leaves were phloridzin and phloretin which accumulated in mM concentrations. Untreated and treated stems and roots contained only phloridzin as the major detectable metabolite during the course of the investigation. The accumulation of phloridzin and phloretin was not developmentally regulated, since they were present in both young and old leaves, and also in the intercellular washings of both scab-susceptible and scab-resistant plants. The major metabolites of both McIntosh and Liberty fruits were cinnamyl glucose and p-coumarylquinic acid, which increased 20-fold in Liberty fruit upon yeast extract treatment. The same compounds increased only 2-fold in McIntosh fruits. Minor compounds in the fruits of both cultivars were p-coumaric acid, phloridzin and phloretin, the latter compound being present at the threshold of detection. Biphenyl and dibenzofuran compounds, the major metabolites of elicitor treated Liberty cell suspension cultures, could not be detected in the intact plants. These results indicate differential response of plant organs and cell suspension cultures to elicitor treatment or pathogen invasion.

We present evidence that susceptible Arabidopsis plants accelerate their reproductive development and alter their shoot architecture in response to three different pathogen species. We infected 2-week-old Arabidopsis seedlings with two bacterial pathogens, Pseudomonas syringae and Xanthomonas campestris, and an oomycete, Peronospora parasitica. Infection with each of the three pathogens reduced time to flowering and the number of aerial branches on the primary inflorescence. In the absence of competition, P. syringae and P. parasitica infection also increased basal branch development. Flowering time and branch responses were affected by the amount of pathogen present. Large amounts of pathogen caused the most dramatic changes in the number of branches on the primary inflorescence, but small amounts of P. syringae caused the fastest flowering and the production of the most basal branches. RPS2 resistance prevented large changes in development when it prevented visible disease symptoms but not at high pathogen doses and when substantial visible hypersensitive response occurred. These experiments indicate that phylogenetically disparate pathogens cause similar changes in the development of susceptible Arabidopsis. We propose that these changes in flowering time and branch architecture constitute a general developmental response to pathogen infection that may affect tolerance of and/or resistance to disease.

Consumption of locoweeds, legumes endemic in arid western USA, has long been associated with locoism, a disease of ruminant animals. To explore the relationship between fungi associated with locoweed and locoweed toxicity, 11 locoweed populations from various sites in New Mexico were assessed for endophytic fungi. Endophytes were isolated from the leaves, stems, seeds, and flowers of eight populations of the toxic locoweeds Astragalus mollissimus, Oxytropis lambertii, and O. sericea. Fungal cultures grew very slowly and sporadically produced subcylindrical conidia with very dark transverse septa. All cultured endophytes produced the alkaloid swainsonine, which causes locoism. Endophyte-infected locoweed populations produced swainsonine, and the swainsonine level of endophyte strains in vitro was highly correlated with the swainsonine level of their host plant populations. The rDNA ITS from mycelia from four endophyte isolates and beta-tubulin encoding regions from mycelia of 18 fungal endophyte isolates were amplified using PCR and the nucleic acid sequences were analyzed. The nucleic acid sequences of the beta-tubulin encoding regions were essentially identical among all the endophytes regardless of plant genus and locations. Morphological evidence and sequence analysis of the ITS region suggest that the endophytes are most closely related to Embellisia. However, with the paucity of Embellisia species represented in sequence databases, precise taxonomic placement will await further study.

The gene predictions and accompanying functional assignments resulting from the sequencing and annotation of a genome represent hypotheses that can be tested and used to develop a more complete understanding of the organism and its biology. In the model plant Arabidopsis thaliana, we developed a novel approach to constructing whole-genome microarrays based on PCR amplification of the 3' ends of each predicted gene from genomic DNA, and constructed an array representing more than 94% of the predicted genes and pseudogenes on chromosome 2. With this array, we examined various tissues and physiological conditions, providing expression-based validation for 84% of the gene predictions and providing clues as to the functions of many predicted genes. Further, by examining the distribution of expression along the physical chromosome, we were able to identify a region of repressed transcription that may represent a previously undescribed heterochromatic region.

Aims: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts., Methods and Results: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system., Conclusions: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing., Significance and Impact of the Study: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.

The alkaloid lycorine, which is considered to inhibit the last step in ascorbic acid biosynthesis, is produced by Narcissus pseudonarcissus. The growth of two strains (C1 and C3) of Cryptococcus laurentii isolated from root tips of N. pseudonarcissus is inhibited by lycorine, as is the in vivo production of ascorbic acid from L-galactonic acid-gamma-lactone. In contrast, C. laurentii strain C4, isolated from the lycorine-containing bracts of the bulb, was not inhibited by lycorine and did not contain ascorbic acid when cultivated with or without L-galactonic acid-gamma-lactone.

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases.

After screening for siderophore (microbial iron chelator) production, of seven available cultures of soybean (Glycine max L.) root nodule bradyrhizobia, one strain, Bradyrhizobium japonicum NCIM 2746, was selected to confirm its phytopathogenic suppression and soybean growth promotion. Based on chromatographic and spectrophotometric studies, two different siderophores, a hydroxamate type (MW 734) and another catecholate type (MW 1000), were observed. Randomized block design (RBD) analysis of sick-pot studies (soil inoculated with phytopathogens) with an MACS 124 variety of soybean, bacterized with siderophoregenic B. japonicum, showed a marked increase in the percentage of germination, nodulation, chlorophyll, oil, protein content, and number of pods. Field trial study confirmed these pot results, which were evident from enhancement in shoot length, number of branches, chlorophyll content, number of nodules, root length, and number of pods. These results suggest the possibility of exploiting B. japonicum NCIM 2746 as a potential bioinoculant.