A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

Streptomyces urticae sp. nov., isolated from rhizosphere soil of Urtica urens L.

Two novel Gram-stain positive, spore-forming, aerobic actinomycetes, designated NEAU-PCY-1T and NEAU-PCY-2, were isolated from rhizosphere soil of Urtica urens L. collected from Anshan, Liaoning Province, northeast China. The 16S rRNA gene sequence analysis showed that strains NEAU-PCY-1T and NEAU-PCY-2 exhibited 99.8% similarity with each other and are closely related to Streptomyces abietis DSM 42080T (98.2, 98.3%) and Streptomyces fildesensis DSM 41987T (98.0, 98.1%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the two strains formed a cluster with these two closely related species. Moreover, DNA-DNA hybridization results and some phenotypic, physiological and biochemical properties differentiated the two strains from their close relatives in the genus Streptomyces. Based on a polyphasic taxonomy study, strains NEAU-PCY-1T and NEAU-PCY-2 are considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces urticae sp. nov. is proposed, with NEAU-PCY-1T (= DSM 105115T = CCTCC AA 2017015T) as the type strain.

Kribbella pittospori sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of an Australian native apricot tree, Pittosporum angustifolium.

An endophytic actinobacterium, strain PIP 158T, was isolated from the stem of a native apricot tree (Pittosporum angustifolium) collected from the grounds of Flinders University, Adelaide, Australia. As a result of a polyphasic taxonomic study, this strain was identified as a member of the genus Kribbella. This strain was a Gram-stain-positive, aerobic actinobacterium with well-developed substrate mycelia which were non-motile and with hyphae fragmenting into short to elongated rod-like elements. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate in the family Nocardioidaceae, being most closely related to Kribbella sandramycini ATCC 39419T and Kribbella albertanoniae BC640T which share a similarity of 99. 26 and 99.18 % with Kribbella hippodromi S1.4T, respectively. Chemotaxonomic data including cell-wall components, major menaquinones and major fatty acids confirmed the affiliation of strain PIP 158T to the genus Kribbella. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain PIP 158T from the closest related species with validly published names. The name proposed for the novel species is Kribbella pittospori sp. nov. The type strain is PIP 158T (=DSM 23717T=NRRL B-24813T).

Micromonospora is a normal occupant of actinorhizal nodules.

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.

Rational approaches to improving the isolation of endophytic actinobacteria from Australian native trees.

In recent years, new actinobacterial species have been isolated as endophytes of plants and shrubs and are sought after both for their role as potential producers of new drug candidates for the pharmaceutical industry and as biocontrol inoculants for sustainable agriculture. Molecular-based approaches to the study of microbial ecology generally reveal a broader microbial diversity than can be obtained by cultivation methods. This study aimed to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture. To achieve this objective, our study successfully employed rational and holistic approaches including the use of isolation media with low concentrations of nutrients normally available to the microorganism in the plant, plating larger quantities of plant sample, incubating isolation plates for up to 16 weeks, excising colonies when they are visible and choosing Australian endemic trees as the source of the actinobacteria. A hierarchy of polyphasic methods based on culture morphology, amplified 16S rRNA gene restriction analysis and limited sequencing was used to classify all 576 actinobacterial isolates from leaf, stem and root samples of two eucalypts: a Grey Box and Red Gum, a native apricot tree and a native pine tree. The classification revealed that, in addition to 413 Streptomyces spp., isolates belonged to 16 other actinobacterial genera: Actinomadura (two strains), Actinomycetospora (six), Actinopolymorpha (two), Amycolatopsis (six), Gordonia (one), Kribbella (25), Micromonospora (six), Nocardia (ten), Nocardioides (11), Nocardiopsis (one), Nonomuraea (one), Polymorphospora (two), Promicromonospora (51), Pseudonocardia (36), Williamsia (two) and a novel genus Flindersiella (one). In order to prove novelty, 12 strains were characterised fully to the species level based on polyphasic taxonomy. One strain represented a novel genus in the family Nocardioides, and the other 11 strains were accepted as novel species. In summary, the holistic isolation strategies were successful in obtaining significant culturable actinobacterial diversity within Australian native trees that includes rare and novel species.

Fungi associated with Scolytogenes birosimensis (Coleoptera: Curculionidae) infesting Pittosporum tobira.

The bark beetle Scolytogenes birosimensis Niijima is suspected to be involved in the decline of Pittosporum tobira (Thunb. ex Murray) Aiton in the coastal areas of Japan. We isolated fungi from adult S. birosimensis in nine different localities in Japan to assess their potential association and predict their contribution to the success of the beetle. Results from morphological identification of associated fungi showed that the beetle was associated with Fusarium solani and Candida spp. Furthermore, molecular analysis showed that F. solani was most closely related to the plant pathogenic fungus F. solani f. sp. mori. Fungal isolation from surface-sterilized, dissected beetles and scanning electron miscroscope (SEM) observation of the body surface suggested that the associated fungi were carried in the pits on the beetles' elytra. These findings contribute to the understanding of the relationships between S. birosimensis and its associated fungi.

Characterization of 14 raspberry cultivars by solid-phase microextraction and relationship with gray mold susceptibility.

Fourteen raspberry varieties were evaluated over two cropping seasons by solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry. Thirty-six compounds were fully identified, and 10 more compounds were tentatively identified. Despite interannual variability, raspberry varieties can be divided in two main groups on the basis of terpenes and C-13 norisoprenoids. Susceptibility toward Botrytis cinerea , one of the most relevant pathogenic fungi for soft fruits during storage, was also evaluated. On the basis of volatile profiles, it was possible to highlight the relationship between different volatile compounds and resistance to B. cinerea. Volatile profiles and Botrytis susceptibility of the different raspberry varieties evaluated should assist future breeding programs.

Characterization of phosphate-solubilizing fluorescent pseudomonads from the rhizosphere of seabuckthorn growing in the cold deserts of Himalayas.

Isolation and characterization of fluorescent pseudomonads with high phosphate-solubilizing ability is reported from the alkaline and calcium-rich soils with low P availability in the cold desert region of Lahaul and Spiti in the trans-Himalayas of India. Of 216 phosphate-solubilizing isolates, 12 exhibiting high solubilization of tricalcium phosphate (TCP) in NBRIP liquid culture were identified as Pseudomonas trivialis, P. poae, P. fluorescens, and Pseudomonas spp. on the basis of phenotypic features, whole-cell fatty acids methyl ester (FAME) profiles, and 16S rDNA sequencing. These isolates also showed relatively high solubilization of North Carolina rock phosphate (NCRP) in comparison to the solubilization of Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). The solubilization of phosphate substrates by P. trivialis and P. poae is reported for the first time.

Dematiocladium celtidis gen. sp. nov. (Nectriaceae, Hypocreales), a new genus from Celtis leaf litter in Argentina.

A Cylindrocladium-like hyphomycete collected on leaf litter of Celtis tala in Argentina had rDNA sequence data (ITS and LSU) that showed it resides in the Hypocreales, and is a member of the Nectriaceae, closely related to, but distinct from Cylindrocladium. A new genus, Dematiocladium and species, D. celtidis gen. sp. nov. is, therefore, introduced to accommodate this fungus. Based on morphology, it can be distinguished from other conidial hypocrealean genera with hyaline, penicillate conidiophores and cylindrical conidia by lacking stipe extensions and vesicles, and by the presence of brown to dark brown, thick-walled setae.

Isolation and phylogenetic analysis of Xylella fastidiosa from its invasive alternative host, porcelain berry.

A strain of Xylella fastidiosa was isolated from an invasive alternative host species, porcelain berry. Its genetic relationship with strains isolated from a native alternative host, wild grape; a nonnative alternative host, mulberry; and other economically important hosts including cultivated grape, peach, plum, oak, maple and oleander was determined by using sequence analysis of the 16S-23S rRNA intergenic spacer region. Our phylogenetic analysis revealed that the porcelain berry strain is most closely related to the wild grape strain. These two strains are more closely related to the oak, peach, and plum strains than to the mulberry and oleander strains. They are separated from the maple and cultivated grape strains. Our data suggest that suppression of porcelain berry, wild grape, and mulberry in the vicinity of susceptible economically important hosts such as oak, peach, and plum may provide an important control measure for diseases caused by X. fastidiosa.

Inhibitory effects of high pressure and heat on Alicyclobacillus acidoterrestris spores in apple juice.

The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90 degrees C).

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis.

We isolated a strain from hop-resistant Lactobacillus hrevis ABBC45, which had lost a plasmid (pRH45) harboring a putative hop resistance gene, horA. The hop resistance level of this horA-deficient strain, named ABBC45(C), was initially low but gradually induced by repeated growth in media containing progressively increasing levels of hop compounds. Although the hop resistance level was substantially lower than that of the hop-adapted wild type strain, hop-adapted ABBC45(C) (ABBC45(CR)) was still capable of growing in beer, suggesting ABBC45 possesses at least two hop resistance mechanisms. Hop resistance acquired by ABBC45(CR) gradually diminished to the pre-adapted level, when the strain was grown repeatedly in the absence of hop compounds. ABBC45(CR) was found to be cross-resistant to several structurally unrelated drugs, including ethidium bromide, daunomycin and nisin. In addition, ABBC45(CR) was shown to extrude ethidium in an energy-dependent manner, while ABBC45(C) did not show such activity. This indicates that the efflux pump was induced by adaptation to hop compounds. The efflux activity of ethidium was reduced by the addition of hop compounds, suggesting hop compounds are also the substrate of the efflux pump. It was also shown that the efflux activity was completely dissipated with the abolition of proton motive force (PMF). These results, taken together, suggest the hop resistance mechanism of ABBC45(C) is mediated by PMF-dependent multidrug efflux pump.

Control of Penicillium expansum and Botrytis cinerea on apples and pears with the combination of Candida sake and Pantoea agglomerans.

The effectiveness of Candida sake (CPA-1) in combination with Pantoea agglomerans (CPA-2) for controlling Penicillium expansum and Botrytis cinerea on pears and apples was determined. The concentrations tested were 2 x 10(6) and 2 x 10(7) CFU/ml for C. sake and 2 x 10(7) and 8 x 10(7) CFU/ml for P. agglomerans. At room temperature, the two antagonists were combined in proportions of 0 to 100% in 25% increments. At the proportion of 50:50, no rot development was observed in pears, and the greatest control of blue mold in apples was observed at this proportion for all the tested concentrations. Under cold temperature on pears, the highest effectiveness of the mixture was observed when C. sake at 2 x 10(7) CFU/ml was combined with P. agglomerans at 2 x 10(7) or at 8 x 10(7) CFU/ml at the proportion 50:50. Under these conditions, no rot development of blue mold was reported, and gray mold lesion size was reduced by more than 95%. On apples, the mixture of C. sake at 2 x 10(7) CFU/ml and P. agglomerans at 8 x 10(7) CFU/ml at the proportion 50:50 reduced blue and gray mold incidence by 90%. Populations of the two antagonists had the same growth pattern at 20 degrees C when they were applied individually or in combination, but the population level was always higher when they grew alone. In contrast, at 1 degrees C, the population of both antagonists in combination formed a stable community with the same levels as individual application during the first 30 days; after that, C. sake dominated, and P. agglomerans decreased on apples and pears. At both temperatures, the maximum population level of C. sake was observed in apples, and themaximum population level of P. agglomerans was observed in pears.

A study of U.S. orchards to identify potential sources of Escherichia coli O157:H7.

The association of unpasteurized apple cider with Escherichia coli O157:H7 foodborne illness has led to increased interest in potential reservoirs of this pathogen in the orchard. Fourteen U.S. orchards were surveyed in autumn 1999 to determine the incidence and prevalence of E. coli O157:H7, E. coli, total aerobic microflora, and yeasts and molds. Fruit samples (n = 63) (eight apple and two pear varieties) and soil, water, and fecal samples were collected. Samples were plated on (i) tryptic soy agar for total mesophilic aerobic count, (ii) E. coli and coliform Petrifilm for total coliforms and E. coli, and (iii) yeast and mold Petrifilm. Samples positive for coliforms and E. coli were enriched and tested for E. coli O157:H7. Fruit was also tested for internalization of microflora by aseptically removing the core, stem, and calyx areas, and the individual sections were assessed for the categories of microflora listed above. E. coli was detected in soil and water and in 6% of fruit samples (three pear samples and one apple sample), generally collected from areas previously designated as high risk in this study. However, no E. coli O157:H7 was found. Coliforms were found in 74% of fruit samples and were internalized in the cores of 40% of fruit tested. Yeasts and molds were internalized in 96.7% of samples and aerobic bacteria in 89.6%. E. coli was not found to be internalized. Total aerobic counts and total coliforms were higher in dropped and damaged fruit (P < 0.05). Findings suggest that dropped or damaged fruit should not be included in fruit designated for the production of unpasteurized juice or for the fresh or fresh-cut market. In addition, orchards should be located away from potential sources of contamination, such as pastures.

Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing.

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Ascorbic acid enhances destruction of Escherichia coli O157:H7 during home-type drying of apple slices.

Destruction of Escherichia coli O157:H7 was evaluated on inoculated apple slices dehydrated at two temperatures with and without application of predrying treatments. Half-ring slices (0.6 cm thick) of peeled and cored Gala apples were inoculated by immersion for 30 min in a four-strain composite inoculum of E. coli O157:H7. The inoculated slices (8.7 to 9.4 log CFU/g) either received no predrying treatment (control), were soaked for 15 min in a 3.4% ascorbic acid solution, or were steam blanched for 3 min at 88 degrees C immediately prior to drying at 57.2 or 62.8 degrees C for up to 6 h. Samples were plated on tryptic soy (TSA) and sorbitol MacConkey (SMAC) agar media for direct enumeration of surviving bacterial populations. Steam blanching changed initial inoculation levels by +0.3 to -0.7 log CFU/g, while immersion in the ascorbic acid solution reduced the inoculation levels by 1.4 to 1.6 log CFU/g. Dehydration of control samples for 6 h reduced mean bacterial populations by 2.9 log CFU/g (TSA or SMAC) at 57.2 degrees C and by 3.3 (SMAC) and 3.5 (TSA) log CFU/g at 62.8 degrees C. Mean decreases from initial inoculum levels for steam-blanched slices after 6 h of drying were 2.1 (SMAC) and 2.0 (TSA) log CFU/g at 57.2 degrees C, and 3.6 (TSA or SMAC) log CFU/g at 62.8 degrees C. In contrast, initial bacterial populations on ascorbic acid-pretreated apple slices declined by 5.0 (SMAC) and 5.1 (TSA) log CFU/g after 3 h of dehydration at 57.2 degrees C, and by 7.3 (SMAC) and 6.9 (TSA) log CFU/g after 3 h at 62.8 degrees C. Reductions on slices treated with ascorbic acid were in the range of 8.0 to 8.3 log CFU/g after 6 h of drying, irrespective of drying temperature or agar medium used. The results of immersing apple slices in a 3.4% ascorbic acid solution for 15 min prior to drying indicate that a predrying treatment enhances the destruction of E. coli O157:H7 on home-dried apple products.

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine.

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.

The effects of freezing and thawing on the survival of Escherichia coli O157:H7 in apple juice.

Unpasteurized apple juice, adjusted to pH 3.6 to 7.0 was inoculated (10(7) CFU/ml) with single strains of E. coli O157:H7 to evaluate the effect of frozen storage on the viability of this organism. Samples were stored under frozen conditions (-20+/-2 degrees C) for up to 16 days. Cell populations were determined at regular intervals by plating onto tryptic soy agar with added pyruvate (TSAP) or onto sorbitol MacConkey agar (SMA). Populations in the neutralized juice remained unchanged during frozen storage. Populations in non-neutralized juice decreased by 1-3 log10 CFU/ml depending on the strain tested and the pH of the juice. The greatest population decrease was observed with the first freeze/thaw cycle of frozen storage (24 h) and a slow decline in survival occurred thereafter. Injury was observed after 2 weeks of storage when juice pH was at or below pH 4.2. When samples were subjected to multiple freeze/thaw cycles, loss of viability and injury increased with each freeze/thaw cycle.

Reduction in levels of Escherichia coli O157:H7 in apple cider by pulsed electric fields.

Many studies have demonstrated that high voltage pulsed electric field (PEF) treatment has lethal effects on microorganisms including Escherichia coli O157:H7; however, the survival of this pathogen through the PEF treatment is not fully understood. Fresh apple cider samples inoculated with E. coli O157:H7 strain EC920026 were treated with 10, 20, and 30 instant charge reversal pulses at electric field strengths of 60, 70, and 80 kV/cm, at 20, 30, and 42 degrees C. To accurately evaluate the lethality of apple cider processing steps, counts were determined on tryptic soy agar (TSA) and sorbitol MacConkey agar (SMA) to estimate the number of injured and uninjured E. coli O157:H7 cells after PEF treatment. Cell death increased significantly with increased temperatures and electric field strengths. A maximum of 5.35-log10 CFU/ml (P < 0.05) reduction in cell population was achieved in samples treated with 30 pulses and 80 kV/cm at 42 degrees C. Cell injury measured by the difference between TSA and SMA counts was found to be insignificant (P > 0.05). Under extreme conditions, a 5.91-log10 CFU/ml reduction in cell population was accomplished when treating samples with 10 pulses and 90 kV/cm at 42 degrees C. PEF treatment, when combined with the addition of cinnamon or nisin, triggered cell death, resulting in a reduction in E. coli O157:H7 count of 6 to 8 log10 CFU/ml. Overall, the combination of PEF and heat treatment was demonstrated to be an effective pasteurization technique by sufficiently reducing the number of viable E. coli O157:H7 cells in fresh apple cider to meet U.S. Federal Drug Administration recommendations.