Differential Effects of Lithium Chloride on In Vitro Growth of Clavibacter michiganense subsp. nebraskense Depending upon Inoculum Source

The bacterium Clavibacter michiganense subsp. nebraskense (Corynebacterium michiganense subsp. nebraskense) was grown in broth cultures and inoculated into corn plants. The plating efficiency of cells from broth cultures was essentially the same on nutrient broth-yeast extract and the semiselective medium for this bacterium, CNS. However, when cells were isolated from Goss bacterial wilt- and blight-infected corn, very few were recovered on CNS compared with the amount recovered on nutrient broth-yeast extract agar. When lithium chloride was omitted from the CNS, recoveries from infected corn were nearly the same as on nutrient broth-yeast extract agar. No other ingredient of CNS was inhibitory, nor did substitution of other salts for lithium chloride cause equal inhibition. The amount of inhibition was proportional to lithium chloride concentration. The inhibition by lithium chloride occurred with several strains of the bacterium isolated from one corn cultivar and with one of the strains recovered from three different cultivars of infected corn.     

Induced androgenesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.). III. Characterization of the regenerants

We present data on the morphological, cytological, biochemical and genetic characteristics of tomato regenerants obtained through anther culture. As a result of induced androgenesis, more than 6,000 rooted regenerants were developed that differed both from the donor plants and among each other with respect to habitus and leaf, flower and inflorescence morphology. Cytological analysis revealed a great variability in chromosome number in the cells of the regenerated plants. While most of the regenerants were mixoploid, the majority of the cells had a haploid chromosome number. R₁ and R₂ progenies were tested for their resistance to Clavibacter michiganense subsp. michiganense (Cmm 7). Some of the regenerants were resistant to the pathogen. A biochemical analysis of fruit from R₃ and R₄ plants showed a higher content of dry matter, sugars and vitamin C in the regenerant plants obtained from the hybrids than in those from the cultivars and control plants. The values of the parameters of hybrid regenerants grown in the greenhouse were about 1.5-fold higher than those of the hybrid regenerants grown in the field, and this trend is clearly expressed in all of the hybrid regenerants. The results obtained suggest that induced androgenesis and gametoclonal variation may be used as an additional tool to create a large range of new forms. The application of the latter in breeding programs would accelerate the development of tomato lines and varieties that would be more productive, disease-resistant, highly nutritive and flavour-acceptable.Abbreviations BAP N⁶-Benzylaminopurine - Cmm Clavibacter michiganense subsp. michiganense - cfu Colony-forming units - GA ₃ Gibberellic acid - IBA Indole-3-butyric acid - ms Male sterility - PDA Potato dextrose agar Communicated by H. Lörz     

Anion channel forming activity from the plant pathogenic bacteriumClavibacter michiganense ssp. nebraskense

Summary The plant pathogenic bacteriumClavibacter michiganense ssp. nebraskense secretes an anion channel forming activity (CFA) into the culture fluid. The CFA inserts spontaneously into planar lipid membranes when culture fluid of this species is added to the aqueous phase of the bilayer chamber. The channels formed are highly anion selective. The conductance decreases for larger anions (Cl^–>SCN^–>SO ₄ ^2– ) and is practically zero for gluconate. The channels show a unique voltage dependence : (i) The single-channel conductance increases linearly with voltage up to 200 mV saturating at 250 mV with 25±1 pS (300mm KCl). The channel is closed at negative voltage relative to the side of insertion (diode-typeI–V curve). (ii) The average number of open channels also increases with voltage. The Poisson distribution of channel numbers indicates independent opening of the channels.Channel activity can be abolished by protease treatment of the planar bilayer. The channels can be blocked by indanyloxyacetic acid (IAA-94) and by pH>10. The CFA was purified yielding one major band on the SDS gel with a relative molecular mass of 65,000. The putative involvement of the CFA in the toxicity of this plant pathogen is discussed and compared to other toxins like colicins and to the diphtheria toxin group.     

Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR

Aim

Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously.

Methods and Results

Test‐systems for real‐time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier.

Conclusions

Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA‐ and DNA‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg).

Significance and Impact of the Study

The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.     

Genetic transformation of cotton with a harpin-encoding gene <Emphasis Type="Italic">hpa</Emphasis><Subscript><Emphasis Type="Italic">Xoo</Emphasis></Subscript> confers an enhanced defense response against different pathogens through a priming mechanism

Background  

The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful.     

Transmission of Bacterium Corynebacterium michiganense pv. michiganense by Seeds

The results of experiments on transmission of Corynebacterium michiganense pv. michiganense showed that the spread of the bacterium by artificially infested seeds varies, but it was confirmed that from artificially infested seeds few diseased plants can be grown. The results depend on the environmental conditions during experimentation and the method of assaying the seedlings. High relative humidity favours the spread of the pathogen. The use of selective media helped the detection ofthe pathogen in seemingly healthy plants. It was proved that host can have population of C. michiganense pv. michiganense in a resident phase. It is assumed that when conditions are favorable the pathogen multiplies and cause disease.     

Circulating regulatory T cells are reduced in obesity and may identify subjects at increased metabolic and cardiovascular risk

Objective:

Reduced numbers of regulatory T (T_reg) cells have been observed in visceral adipose tissue of obese mice and humans. However, it is unknown whether human obesity affects circulating Treg cells and whether their number is associated with markers of systemic inflammation or glucose intolerance.

Design and Methods:

Peripheral blood mononuclear cells were isolated from venous blood of obese (BMI ≥ 27 kg/m²; n = 30) and nonobese (BMI ≥ 27 kg/m²; n = 13) individuals and analyzed using flow cytometry for the expression of CD4, CD25, and Foxp3.

Results:

Reduced circulating T_reg‐cell numbers were detected in obese compared with nonobese study participants (P = 0.038). Circulating CD4⁺CD25⁺CD127^?Foxp3 T_reg cells inversely correlated with body weight (P = 0.009), BMI (P = 0.004) and plasma leptin levels (P = 0.004) and were reduced in subjects with hsCRP ≥ 3.0 mg/L (P = 0.034) or HbA1c ≥ 5.5% (P < 0.005). Receiver operating characteristic curve analysis revealed a cutoff of circulating Treg cells < 1.06% to be predictive for hsCRP levels ≥ 3.0 mg/L, and logistic regression showed that the risk of having hsCRP levels ≥ 3.0 mg/L was increased 9.6‐fold (P = 0.008), if T_reg cells were below this threshold. The T_reg cutoff for HbA1c levels ≥ 5.5% was 0.73%, and this cutoff also predicted an increased risk of having elevated levels of both hsCRP and HbA1c, if only obese subjects were examined.

Conclusion:

Our findings thus reveal an association between circulating T_reg cells and measures of adiposity, inflammation, and glucose intolerance. Although further prospective studies are needed, we present data suggesting that the determination of T_reg cells might be useful to identify obese subjects at increased risk of developing cardiovascular and/or metabolic complications.

     

Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage

Background  

MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, BRCA1 ^fl/fl ;p53 ^+/-;MMTV-cre knock-out mice and the p53 ^fl/fl ;MMTV-cre transplant model.     

Kinetics of metal toxicity in plant roots and its effects on root morphology

Aims

We evaluated the efficacy of biochar application for suppressing bacterial wilt of tomato and identified the potential underlying mechanisms involved in the disease control.

Methods

We measured the impact of two different sized biochar (53–120 μm and 380–830 μm) on bacterial wilt incidence in a greenhouse experiment. The efficiency of different sized biochar for the adsorption of tomato root exudates and the pathogen was further examined in vitro. We also quantified the effects of biochar and tomato root exudates on two pathogen virulence factors, chemotaxis, swarming motility and examined the effect of biochar on pathogen root colonization.

Results

Fine biochar application (3%; w:w) significantly decreased the bacterial wilt incidence by 19.9%. Biochar with different particle size had similar adsorption capacity for root exudates, while fine biochar was efficient (91%) in pathogen adsorption. Root exudates and fine biochar increased the chemotaxis ability of pathogen, while fine biochar reduced pathogen swarming motility and rhizosphere colonization.

Conclusions

Application of fine biochar can significantly decreased bacterial wilt incidence. This was mechanistically explained by biochar ability to 1) adsorb pathogen directly and indirectly via adsorption of root exudates (based on pathogen chemotaxis) and to 2) directly suppress pathogen swarming motility and subsequent root colonization.

     

Contribution of Cd-EDTA complexes to cadmium uptake by maize: a modelling approach

Background and aims

Chelant-enhanced phytoextraction has given variable and often unexplained experimental results. This work was carried out to better understand the mechanisms of Cd plant uptake in the presence of EDTA and to evaluate the contributions of Cd-EDTA complexes to the uptake.

Method

A 1-D mechanistic model was implemented, which described the free Cd²⁺ root absorption, the dissociation and the direct absorption of the Cd-EDTA complexes. It was used to explain Cd uptake by maize in hydroponics and in soil.

Results

In hydroponics, the addition of EDTA caused a decrease in Cd uptake by maize, particularly when the ratio of total EDTA ([EDTA] _T ) to total Cd ([Cd] _T ) was greater than 1. At [Cd] _T = 1 μM, when [EDTA] _T /[Cd] _T < 1, the model indicated that Cd uptake was predominantly due to the absorption of free Cd²⁺, whose pool was replenished by the dissociation of Cd-EDTA. When [EDTA] _T /[Cd] _T > 1, the low Cd uptake was mostly due to Cd-EDTA absorption. In soil spiked with 5 mg Cd kg^?1, Cd uptake was not affected by the various EDTA additions, because of the buffering capacity of the soil solid phase.

Conclusions

Addition of EDTA to soil increases Cd solubility but dissociation of Cd-EDTA limits the availability of the free Cd²⁺ at the root surface, which finally reduces the plant uptake of the metal.     

Sciarid and shore flies as aerial vectors of Fusarium oxysporum f. sp. cucumerinum in greenhouse cucumbers

The options for managing Fusarium wilt in greenhouse cucumbers are limited by our poor understanding of the modes of survival and dissemination of the pathogen. This study uses a specific quantitative real‐time PCR assay for Fusarium oxysporum f. sp. cucumerinum to investigate the significance of flying insects as aerial vectors of the pathogen in a commercial cucumber greenhouse. Shore flies were more frequently detected (35.5%) carrying F. oxysporum f. sp. cucumerinum than sciarids (25%), with both species carrying between 1 × 10² and 1 × 10⁶ pathogen genome copies/individual. Sciarid and shore flies acquired F. oxysporum f. sp. cucumerinum following exposures to agar cultures of the pathogen of up to 94 h. Light microscopy revealed that spores were carried externally on the bodies of the adult flies. The ability of adult sciarid flies to vector the pathogen from peat‐grown diseased cucumber plants and infect healthy cucumber plants was demonstrated in a caged glasshouse trial. An inoculum density trial showed that vascular wilt disease was initiated after inoculation of peat‐grown seedlings with as few as 1000 conidia. We conclude that sciarid and shore flies play significant roles as vectors of F. oxysporum f. sp. cucumerinum in greenhouse cucumbers and need to be recognized in developing integrated crop management strategies.     

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

Background  

The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.     

Length to weight ratio of four fish species in estuary areas in the Northeast region of Brazil

The length to weight ratio (LWR) was used to estimate populations of four fish species sampled on the Amazon coast of the state of Maranhão, Brazil. The fishes were sampled in 2018 and 2019 by artisanal coastal fishing, using gillnets (0.20–0.60 mm). The parameters a and b of the equation W_T = a L_T^b were estimated. The obtained LWRs were W_T = 0.06LT^2.93, (r²) = .989 for Oligoplites saliens; W_T = 0.061L_T^2.57, (r²) = .982 for Peprilus crenulatus; W_T = 0.014L_T^2.80, (r²) = .985 for Bagre bagre; and W_T = 0.035L_T^3.21, (r²) = .981 for Nebris microps.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.     

Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L.) enhances tolerance to verticillium wilt

Background

Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored.

Methodology/Principal Findings

In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T₃ generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T₁–T₃ generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines''s 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD).

Conclusion/Significance

Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.     

Sources of microorganisms in pozol,a traditional Mexican fermented maize dough

Freshly prepared pozol, a traditional Mexican fermented maize dough, contained (c.f.u./g wet wt): lactic acid bacteria, 10⁴ to 10⁶; aerobic mesophiles, 10⁴ to 10⁵; Enterobacteriaceae, 10² to 10³; yeasts, 10² to 10⁴; and mould propagules, <10³. After 30 h at 28°C the numbers were, respectively: 10⁹, 7×10⁶, 5×10⁵, 10⁶ and 10⁴. Soaking alkali-treated grains overnight allowed lactic acid bacteria, aerobic mesophiles and Enterobacteriaceae to grow and these then constituted the primary microbial flora of the pozol dough. Grinding in a commercial mill inoculated the dough with lactic acid bacteria, aerobic mesophiles, Enterobacteriaceae and yeasts. Other processing stages, including the nature of the surface upon which the balls were made, handling of the dough, and air, contributed only minor numbers of microbes compared with the two major sources, soaking and grinding. The pH of pozol fell from an initial value of 7.3 to 4.6 after 30 h incubation at 28°C. The numbers of Enterobacteriaceae and other aerobic mesophilic bacteria remained constant between 11 and 30 h incubation and there was no evidence of the acidic conditions having any lethal effects on these organisms.     

Ventilatory physiology of the numbat (<Emphasis Type="Italic">Myrmecobius fasciatus</Emphasis>)

This study examines the ventilatory physiology of the numbat (Myrmecobius fasciatus), a small to medium-sized (550 g) termitivorous marsupial. Ventilatory parameters at thermoneutrality reflect the slightly low (83% of predicted) basal metabolic rate of the numbat, with ventilation frequency (ƒ_R; 30.6±3.65 breaths min^–1), tidal volume [V_T; 6.0±0.66 ml at body temperature and pressure, saturated (BTPS)] and consequently minute volume (V_I; 117.7±15.22 ml min^–1; BTPS) all being 80–87% of that expected for a marsupial of similar body mass. Oxygen extraction was 27.7±1.37% in the thermoneutral zone. As is typical of marsupials, numbats accommodated increased oxygen consumption rates at ambient temperatures (T _a) below the thermoneutral zone by increasing minute volume (up to 411.2±43.98 ml min^–1; BTPS at T _a=10 °C) rather than oxygen extraction. Minute volume at 10 °C increased more by changes in ventilation frequency (up to 45.5±4.85 breaths min^–1) than tidal volume (9.4±1.03 ml, BTPS), as is also typical for a small-medium sized marsupial.Abbreviations BMR basal metabolic rate - BTPS body temperature and pressure, saturated - EO ₂ oxygen extraction - ƒ _R ventilation frequency - STPD standard temperature and pressure, dry - T _a ambient temperature - T _b body temperature - TNZ thermoneutral zone - V _I minute volume - V _T tidal volume - O ₂ oxygen consumption rate Communicated by I.D. Hume     

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.