Osmotic shock tolerance and membrane fluidity of cold-adapted Cryptococcus flavescens OH 182.9, previously reported as C. nodaensis, a biocontrol agent of Fusarium head blight

Cryptococcus flavescens (previously reported as C. nodaensis), a biological control agent of Fusarium head blight, has been previously shown to have improved desiccation tolerance after cold adaptation. The goal of the current study was to determine the effect of cold adaptation on the physicochemical properties of C. flavescens that may be responsible for its improved desiccation tolerance. The results show that cold adaptation improves liquid hyperosmotic shock tolerance and alters the temperature dependence of osmotic shock tolerance. Fluorescence anisotropy was used to characterize differences in the membrane fluidity of C. flavescens with and without cold adaptation. Force curves from atomic force microscopy showed a significant increase in the cell wall spring constant after cold adaptation. Cold adaptation of C. flavescens during culturing was shown to produce smaller cells and produced a trend towards higher CFU yields. These results suggest that cold adaptation significantly alters the membrane properties of C. flavescens and may be an effective method of improving the desiccation tolerance of microorganisms. In addition, we provide information on the correct naming of the isolate as C. flavescens.     

Population dynamics of the Fusarium head blight biocontrol agent Cryptococcus flavescens OH 182.9 on wheat anthers and heads

Cryptococcus flavescens OH 182.9 (NRRL Y-30216) reduces Fusarium head blight (FHB) incited by Fusarium graminearum and deoxynivalenol (DON) contamination of grain. Yet little is known about the population dynamics of OH 182.9 on wheat heads and anthers. Biomass of OH 182.9 was produced in liquid culture and applied to greenhouse and field grown wheat prior to and during early anthesis. In greenhouse studies, populations of OH 182.9 were similar on anthers for heads inoculated before (Feekes 10.5) or early in flowering (Feekes 10.5.1) but were 1–3 log units lower in Feekes 10.5 inoculated wheat after 8–10 days. In greenhouse and field studies, OH 182.9 colonized anthers inside florets prior to anthesis. In the field, populations of OH 182.9 on anthers increased or, less frequently, remained stable through 12 days, regardless of application time and peaked at 1–2 log units higher than in the greenhouse. Strain OH 182.9 reduced FHB severity (P < 0.05, FPLSD) but not other disease parameters in the same field study. Application of OH 182.9 at split boot (Feekes 10.1) or Feekes 10.5.1 resulted in higher populations on spikelets treated at flowering on a CFU/g fresh weight tissue basis and as a percentage of the total recoverable microbial population in one of two field studies. Scanning electron microscopy revealed cells of OH 182.9 in microcolonies, groups of several cells and as individual cells, most frequently on the abaxial surfaces of glume and lemma tissues and near the apex of palea tissues. The survival of yeast OH 182.9 on anthers and wheat heads for 12 days and more suggests the strain has the potential to reduce late kernel infections by F. graminearum that can increase DON.     

Utilization of chemical inducers of resistance and Cryptococcus flavescens OH 182.9 to reduce Fusarium head blight under greenhouse conditions

Four chemicals [salicylic acid (SA), sodium salt of salicylic acid (NaSA), isonicotinic acid (INA), and DL-β-amino-n-butyric acid (BABA)] and the yeast antagonist Cryptococcus flavescens (=C. nodaensis nomen nudum) OH 182.9 were evaluated separately or together for the ability to reduce Fusarium head blight (FHB) of wheat in the greenhouse. When sprayed onto wheat heads at 3 days prior to pathogen challenge with Gibberella zeae, NaSA and INA at 10 mM significantly reduced FHB severity compared to the non-treated disease control. Applied at concentrations of 1 and 5 mM at 3 days before pathogen challenge, NaSA or INA in combination with OH 182.9 did not significantly reduce FHB severity compared to either treatment alone, though the lowest disease severity values frequently were associated with the combination treatments. When sprayed onto wheat heads just beginning to emerge from boot at 10 days prior to pathogen inoculation, NaSA, INA, and BABA at 1 mM significantly reduced FHB severity indicating that induced systemic resistance was at least partially responsible for the reduction of FHB disease. Induced FHB resistance was achieved by treating wheat with INA at concentrations as low as 0.1 mM. In only one instance was 100-kernel weight affected by any chemical or combination of chemicals with OH 182.9 treatment. Data from our studies in the greenhouse suggest that chemical inducers can induce resistance in wheat against FHB, and that further efforts are warranted to explore the potential of improved control of FHB disease by incorporating chemical inducers with the FHB biocontrol agent OH 182.9.     

Osmotic stress adaptation,compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.     

Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita,a biocontrol agent for slugs

The rhabditid nematode Phasmarhabditis hermaphrodita (a parasite capable of killing pest slugs) was grown in vitro, in association with a mixed bacterial flora on foam chips impregnated with a kidney‐based nutrient medium in aerated bags, to provide sufficient numbers for laboratory and field experiments. The feasibility of producing nematodes in liquid culture was investigated using 250 ml flasks. Baffled flasks containing 25 or 50 ml or liquid were found to be better than baffled flasks containing 100 ml or unbaffled flasks. Inoculum densities ranging from 50 to 330 ml^‐1 did not affect final yield. Dauer larvae in aerated water died rapidly at temperatures of 26–35°C. Survival was progressively better at 22°C and 15°C, and best at 5°C or 10°C.     

Saturation and comparative mapping of a major Fusarium head blight resistance QTL in tetraploid wheat

Fusarium head blight (FHB) is a devastating disease of cultivated wheat worldwide. Partial resistance to FHB has been identified in common wheat (Triticum aestivum L.). However, sources of effective FHB resistance have not been found in durum wheat (T. turgidum L. var. durum). A major FHB resistance quantitative trait loci (QTL), Qfhs.ndsu-3AS, was identified on chromosome 3A of T. dicoccoides, a wild relative of durum wheat. Here, we saturated the genomic region containing the QTL using EST-derived target region amplified polymorphism (TRAP), sequence tagged site (STS), and simple sequence repeat (SSR) markers. A total of 45 new molecular marker loci were detected on chromosome 3A and the resulting linkage map consisted of 55 markers spanning a genetic distance of 277.2 cM. Qfhs.ndsu-3AS was positioned within a chromosomal interval of 11.5 cM and is flanked by the TRAP marker loci, Xfcp401 and Xfcp397.2. The average map distance between the marker loci within this QTL region was reduced from 4.9 cM in the previous study to 3.5 cM in the present study. Comparative mapping indicated that Qfhs.ndsu-3AS is not homoeologous to Qfhs.ndsu-3BS, a major FHB QTL derived from the common wheat cultivar Sumai 3. These results facilitate our efforts toward map-based cloning of Qfhs.ndsu-3AS and utilization of this QTL in durum wheat breeding via marker-assisted selection.     

Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight

Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU(gf.w.)(-1), which in turn decreased progressively to 4-5 log CFU(gf.w.)(-1) after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU(gf.w.)(-1), thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Genome sequencing and analysis of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> strain 34-9, a biocontrol agent against postharvest pathogens in citrus

Kloeckera apiculata, as the anamorphic state of Hanseniaspora uvarum from the Ascomycota phylum, plays an important role in the inhibition of fungal diseases in plants and spontaneous wine fermentation. This study was performed to sequence and analyze the whole genome of K. apiculata strain 34-9; This analysis provides further genomic features and assists functional research. The complete genome was determined using an Illumina HiSeq 2000 system applying paired-end and mate-pair methods to construct four reads libraries. The data assembly of all the reads resulted in a total genome size of 8.1 Mb, including 106 contigs, which were assembled into 41 scaffolds with a 31.95 % G+C content and a 234X sequence coverage. The performance of the gene prediction and functional annotation revealed that 2724 of 3786 protein-coding genes matched the KOG database, and 1127 genes were classified into GO categories. Further genome features analyses found 1066 microsatellite sites, 71 tRNAs, 3 rRNAs and 3 microRNAs in the genomic DNA. A prediction of the metabolic pathways identified potentially crucial genes for explaining the phenylalanine pathway involved in biocontrol. Comparisons with the typical yeasts Lachancea thermotolerans, Kluyveromyces lactis and Saccharomyces cerevisiae revealed the particularity and difference of K. apiculata strain 34-9. The genome alignments among Hanseniaspora vineae T02/19AF, K. apiculata DSM 2768 and 34-9 indicated numerous homologous regions distributed over the genomes between strain DSM2768 and 34-9. A SSR analysis identified that mono- and tri- nucleotide repeat types were more abundant in all six types, likely affecting the evolution of K. apiculata.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Complex microcolinearity among wheat, rice, and barley revealed by fine mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat

A major quantitative trait locus (QTL), Qfhs.ndsu-3BS, for resistance to Fusarium head blight (FHB) in wheat has been identified and verified by several research groups. The objectives of this study were to construct a fine genetic map of this QTL region and to examine microcolinearity in the QTL region among wheat, rice, and barley. Two simple sequence repeat (SSR) markers (Xgwm533 and Xgwm493) flanking this QTL were used to screen for recombinants in a population of 3,156 plants derived from a single F₇ plant heterozygous for the Qfhs.ndsu-3BS region. A total of 382 recombinants were identified, and they were genotyped with two more SSR markers and eight sequence-tagged site (STS) markers. A fine genetic map of the Qfhs.ndsu-3BS region was constructed and spanned 6.3 cM. Based on replicated evaluations of homozygous recombinant lines for Type II FHB resistance, Qfhs.ndsu-3BS, redesignated as Fhb1, was placed into a 1.2-cM marker interval flanked by STS3B-189 and STS3B-206. Primers of STS markers were designed from wheat expressed sequence tags homologous to each of six barley genes expected to be located near this QTL region. A comparison of the wheat fine genetic map and physical maps of rice and barley revealed inversions and insertions/deletions. This suggests a complex microcolinearity among wheat, rice, and barley in this QTL region.     

Survival of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> during heating,drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20g sucrose l^–1or 12.5g monosodium glutamate l^–1. No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55°C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%. Revisions requested 4 November 2004; Revisions received 13 December 2004     

Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum,F. graminearum and F. nivale using a multiplicative model for interaction

To determine whether resistance to Fusarium head blight in winter wheat is horizontal and non-species specific, 25 genotypes from five European countries were tested at six locations across Europe in the years 1990, 1991, and 1992. The five genotypes from each country had to cover the range from resistant to susceptible. The locations involved were Wageningen, Vienna, Rennes, Hohenheim, Oberer Lindenhof, and Szeged. In total, 17 local strains of Fusarium culmorum, F. graminearum, and F. nivale were used for experimental inoculation. One strain, F. culmorum IPO 39-01, was used at all locations. Best linear unbiased predictions (BLUPs) for the head blight ratings of the genotypes were formed within each particular location for each combination of year and strain. The BLUPs over all locations were collected in a genotype-by environment table in which the genotypic dimension consisted of the 25 genotypes, while the environmental dimension was made up of 59 year-by-strain-by-location combinations. A multiplicative model was fitted to the genotype by-environment interaction in this table. The inverses of the variances of the genotype-by-environment BLUPs were used as weights. Interactions between genotypes and environments were written as sums of products between genotypic scores and environmental scores. After correction for year-by-location influence very little variation in environmental scores could be ascribed to differences between strains. This provided the basis for the conclusion that the resistance to Fusarium head blight in winter wheat was of the horizontal and non-species specific type. There was no indication for any geographical pattern in virulence genes. Any reasonable aggressive strain, a F. culmorum strain for the cool climates and a F. graminearum strain for the warmer humid areas, should be satisfactory for screening purposes.     

Evaluation of manometric temperature measurement (MTM), a process analytical technology tool in freeze drying, part III: Heat and mass transfer measurement

This article evaluates the procedures for determining the vial heat transfer coefficient and the extent of primary drying through manometric temperature measurement (MTM). The vial heat transfer coefficients (K_v) were calculated from the MTM-determined temperature and resistance and compared with K_v values determined by a gravimetric method. The differences between the MTM vial heat transfer coefficients and the gravimetric values are large at low shelf temperature but smaller when higher shelf temperatures were used. The differences also became smaller at higher chamber pressure and smaller when higher resistance materials were being freeze-dried. In all cases, using thermal shields greatly improved the accuracy of the MTM K_v measurement. With use of thermal shields, the thickness of the frozen layer calculated from MTM is in good agreement with values obtained gravimetrically. The heat transfer coefficient “error” is largely a direct result of the error in the dry layer resistance (ie, MTM-determined resistance is too low). This problem can be minimized if thermal shields are used for freeze-drying. With suitable use of thermal shields, accurate K_v values are obtained by MTM; thus allowing accurate calculations of heat and mass flow rates. The extent of primary drying can be monitored by real-time calculation of the amount of remaining ice using MTM data, thus providing a process analytical tool that greatly improves the freeze-drying process design and control.     

Effects of local vegetation and plantation age for the parasitoid Asecodes mento– a biocontrol agent in organic strawberry fields

Abstract The parasitoid Asecodes mento (Walker, 1839) (Hymenoptera: Eulophidae) is the most important biocontrol agent of the strawberry leaf beetle Galerucella tenella (L.) (Coleoptera: Chrysomelidae) in northern Europe. Here, I investigated whether natural parasitism in organic strawberry plantations was affected by the presence of the alternative host plant meadowsweet (Filipendula ulmaria), and whether parasitism rates differed between plantations of different ages (6 to 79 years). I also investigated whether parasitoid brood size, body size and sex ratio differed between the two host plants in the field. Parasitism was very low (0%) in newly established plantations and increased to a plateau (～40%) in fields where strawberries had been grown for approximately 20 years or longer. Such an extended colonization process is unacceptable for commercial growers. It would thus be desirable to find a method to catalyze parasitoid population buildup in young plantations. Parasitoid brood sizes were larger in beetles collected from meadowsweet, while body size and sex ratio did not differ between parasitoids collected from the two plants. These findings suggest that meadowsweet can export parasitoids to neighboring strawberry fields. Although this is a possibility, I did not find any significant differences in parasitism rates between isolated strawberry fields and fields adjacent to meadowsweet stands, indicating that effects of local vegetation are small on parasitism rates. Releasing parasitoids in newly established strawberry plantations may be a better strategy for quickly obtaining high parasitism than intercropping with meadowsweet.     

Purification,crystal structure and antimicrobial activity of phenazine‐1‐carboxamide produced by a growth‐promoting biocontrol bacterium,Pseudomonas aeruginosa MML2212

Aim: To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Methods and Results: Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide‐spectrum antimicrobial activity was cultured in Kings’B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two‐step silica‐gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220–222°C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223·3 by mass spectral analysis. Further, it was characterized by IR, ¹H and ¹³C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X‐ray diffraction study and deposited in the Cambridge Crystallographic Data Centre ( http://www.ccde.com.ac.uk ) with the accession no. CCDC 617344 . Conclusion: The crystal compound was undoubtedly identified as phenazine‐1‐carboxamide (PCN) with the empirical formula of C₁₃H₉N₃O. Significance and Impact of the Study: As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.