Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2

Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv. tabaci 153. Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non-transformed controls. The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco 17–38% versus 78%) and the average size of these lesions was 61–81% that of control. The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control). Arabidopsis thaliana transgenic plants inoculated with P. syringae pv. tomato DC3000 also had lower percentages of necrotic lesions (22–38% versus 76%), a reduced average area for each lesion (53–67% of control), and a smaller total lesion area per inoculation (43% of control). These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential.     

Erwinia salicis in Cricket Bat Willows: Rate of Movement of the Bacterium and the Production of Symptoms in Young Trees and Shoots

The lack of movement of Erwinia salicis and the erratic development of symptoms of watermark disease in young rooted leafy cuttings of the cricket bat willow, Salix alba var. caerulea , grown in a controlled environment is described. Erwinia salicis remained in the zone of inoculation for 3 h after inoculation. Fifty-five % of inoculations resulted in the bacterium being isolated on glycerol nutrient agar after 6 months but in the development of none of the classical symptoms of the disease. The maximum penetrations of wood by E. salicis were 24 cm in 24 h in 4·5-year-old wood, 20 cm in 24 h in 2·5-year-old wood and 6 cm in 6 d in 1·5-year-old wood. The pathogen was not isolated from anywhere other than the wood of the original cutting, and did not penetrate into shoots existing before or developing after inoculation, in 6 months. Greatest numbers of E. salicis were present in the 1-cm length of wood including the inoculation site at all time intervals studied. Results using a saprophyte, Serratia marcescens , were similar to those obtained using E. salicis insofar as distribution of the bacteria in the wood with time was concerned. Although E. salicis could still be isolated from test inoculations after 6 months, only 7% of inoculated small trees developed typical watermark symptoms in the wood, though a further 45% showed red leaf symptoms. Movement of the pathogen over longer periods needs study, and older trees need to be artificially inoculated. Young, artificially inoculated cricket bat willow trees, up to 5 years of age are resistant to the systemic spread of E. salicis , and of other bacteria. Symptom expression is very erratic, despite the genetic homogeneity of the host material. The use of very young shoots as propagating material should be tested as a means of controlling the disease.     

Effects of temperature on infection of French bean leaves (Phaseolus vulgaris L.) by lucerne mosaic virus

The effect of temperature on the number of lesions and the time of their appearance was studied by inoculating French bean leaves (Phaseolus vulgaris L. cv. Perli?ka) with lucerne mosaic virus either 24 or 48 h before or, 24 or 48 h after they were exposed to various temperatures. The temperatures tested were 23, 25, 27, 30, 33 and 36° C. Before and after such exposures the plants were kept in a constant temperature of 25° C. By increasing the temperature before inoculation the number of lesions increased in comparison with the control. The optimal temperature for the maximum number of lesions is between 27° and 30° C. There is no significant difference between those experiments when the exposure time was 24 h or 48 h before inoculation. The same temperatures applied for 24 or 48 h after inoculation have a decreasing effect upon the number of lesions formed by LMV on French bean leaves. The decrease is 30 to 75%. In this case the first necrotic local lesions appeared 42 h after inoculation when exposed to higher temperatures above 27° C for 24 h, and 60 h after inoculation when exposed to these temperatures for 48 h. The shape of lesions varied a little in both cases as the pictures show.     

Effect of Temperature on Susceptibility of the Primary Leaves ofPhaseolus vulgaris L. to Red Clover Mottle Virus

Red clover mottle virus isolated in Czechoslovakia was studied in relation to its reaction to varying temperature on primary French bean leaves (Phaseolus vulgaris L.) on which it forms local necrotic lesions. The plants were kept 24 or 48 h before, or 24 or 48 h after inoculation at the temperatures 23, 25, 27, 30, 33 and 36°C. After such exposures the French beans were kept at a constant temperature of 25°C. The lesions were counted at various intervals. In the experiment the optimal temperature for the maximum number of lesions seems to be 36°C 48 h before inoculation. The temperature above 25°C applied 24 h after inoculation seems to have a decreasing effect upon the number of lesions formed by RCMV on primary leaves of French beans and the lesions appeared several hours later, especially at 30, 33 and 36°C. The temperatures 27, 30 and 33°C applied 48 h after inoculation have a further decreasing effect on the number of lesions. The temperature of 36°C applied 48 h after inoculation has an inactivating effect upon RCMV inoculated on French bean leaves and no lesions appeared 5 days after inoculation.     

Detection of a Bacterium Associated with a Leaf Spot Disease of Maize in Brazil

A leaf spot disease of maize occurring in Brazil in the 1980s was described as being caused by the ascomycete Phaeosphaeria maydis (P. Henn) Rane. Payak and Renfro (imperfect form Phyllosticta sp.). Disease symptoms were dark-green water-soaked spots that later became necrotic lesions. There are no reports at present in the literature of re-infection by the fungus under controlled conditions, casting doubt on the true identity of the pathogen. In this study, cytological analyses of lesions at the initial stages did not detect the presence of fungal structures. Bacterial colonies with yellow pigmentation were isolated from the lesions, which reacted positively in hypersensitivity tests in tobacco plants. Maize plants were inoculated with the isolated bacteria. After 72 h incubation in a dew chamber, plants were transferred to a greenhouse, where they remained until evaluation. Typical symptoms of the disease were observed 5–7 days after inoculation of plants, only on treatments inoculated with the bacteria. The bacterium was re-isolated, which suggests its involvement in the initial phases of disease. The bacterium was identified as Pantoea ananas (synonym Erwinia ananas ).     

Biological Control of Fire Blight of Hawthorn (Crataegus monogyna) with Fluorescent Pseudomonas spp. under Protected Conditions

Naturally-occurring epiphytic fluorescent pseudomonads were isolated and characterized in terms of their potential to control fire blight infection of hawthorn, caused by Erwinia amylovora. Preliminary testing and selection of antagonists using an immature pear fruit assay gave some inconsistency in the amount of pathogen suppression on the pear tissue and also in the prediction of biocontrol effectiveness on the intact plant. Selected antagonists provided significant but variable control of fire blight under protected (polythene tunnel and glasshouse) conditions, with isolates HL83 and HL99 giving control of both blossom-blight and shoot-blight. In some cases the degree of control was equal to that of chemical treatments, including agrimycin 17 and experimental bactericides, and was achieved without any numerical advantage of applied control agent over pathogen. The timing of pseudomonad application in relation to pathogen inoculation was found to have a significant effect on the level of control of blossom-blight.     

Immobilization of cells by entrapping in aubasidan

A new technique of immobilization of microbial cells by entrapping in aubasidan, a microbial polysaccharide, was developed. This technique was applied to three cultures: Erwinia aroidea, Pseudomonas sp., and Alcaligenes faecalis, the producers of aspartase and L-aspartate beta-decarboxylase. The new method is effective. After immobilization, microbial cells retained 79-91% of their initial enzymatic activity.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Antagonism of Erwinia herbicola towards Leptosphaeria maculans causing blackleg disease of Brassica napus

Phyllosphere micro-organisms of Brassica napus were isolated and their antagonism against Leptosphaeria maculans , causal agent of blackleg disease, was tested in vitro . In paired culture, Erwinia herbicola was found to be highly antagonistic to L. maculans. Bioassay of the culture filtrate of the bacterium against the test fungus revealed that Erw. herbicola secretes an antifungal substance into the culture medium. This substance was partially thermolabile and markedly reduced the germination and germ tube length of L. maculans . Aqueous bacterial suspensions and cold-sterilized culture filtrates, when applied to the seedlings prior to inoculation, significantly reduced the severity of blackleg disease.     

Alteration of Response of Bean Leaves to Compatible and Incompatible Bacteria

Injection of Red Mexican bean leaves with Pseudomonas phascolicolaRace 2 (compatible, 18 h before P. mors-prunorum or P. phaseolicolaRace 1 (incompatible), or simultaneous inoculation with compatibleand incompatible bacteria (3:1) greatly delayed the appearanceof hypersensitive responses. When compatible bacteria were inoculated12 h or less before incompatible bacteria, or when the ratioof these bacteria was 1:1 or 0.3:1 in simultaneous inoculation,hypersensitive responses did develop. Earlier inoculation withincompatible bacteria delayed the appearance and severity ofdisease symptoms following later inoculation with compatiblebacteria. When selected areas of leaves were inoculated with compatiblebacteria, effects on hypersensitive responses were confinedto these areas when the whole leaf was inoculated later withincompatible bacteria. Inoculation through the upper leaf surfacewith incompatible bacteria did not affect susceptible responseswhen compatible bacteria were inoculated 24 h later throughthe lower surface. Treatment of leaves with heat-killed bacteria or live bacteriain numbers insufficient to cause hypersensitive responses didnot prevent development of these responses following later inoculationwith incompatible bacteria. Use of heat-killed bacteria didsuppress hypersensitive responses in tobacco leaves. Injectionof leaves with cycloheximide (20 p.p.m.) 30 min before inoculationwith incompatible bacteria suppressed leakage of electrolytesand browning of tissues associated with hypersensitive responses.Cycloheximide had little effect on leakage of electrolytes fromleaves inoculated with compatible bacteria or with the developmentof susceptible responses. Exposure of leaves to chloroform vapourdelayed hypersensitive responses by 24 h; treatment with peroxidasehad no effect.     

Effect of Post-infection Application of Fungicides on the Apple Scab Pathogen, Venturia inaequalis (Cke.) Wint.

The Ergosterol-biosynthesis inhibiting (EBI) fungicides had a prolonged inhibitory effect on the growth and sporulation of the fungus, Venturia inaequalis thereby preventing development of typical apple scab symptoms. Atypical lesions in the form of chlorotic or reddish brown flecks had reduced viable sporulation with deleterious effect on subcuticular hyphae when fungicide was applied beyond 144 h but before 170h after inoculation. Myclobutanil (45μg a.i./ml) had the maximum inhibitory effect on sporulation and colony growth while bitertanol (187.50 μg a.i./ml) showed only a temporary inhibition of fungal growth and sporulation. Ultra-structural studies also exhibited different levels of fungitoxicity by different fungicides when applied 144 h after inoculation.     

Sequential pathological studies in goats infected intratracheally withAspergillus fumigatus

Intratracheal inoculation of goats withAspergillus fumigatus spores resulted in the development of characteristic gross and microscopic lesions. The lesions were restricted to lungs and there was no dissemination of infection to other tissues of the body except liver in one goat 16 days after infection. The experiment was continued for 37 days. Gross changes in lungs were observed up to the 24th day post-infection. The lesions, in general, included congestion and oedema in the first 6 days followed by the development of varying greyish-white nodules in the lungs. Microscopic changes consisted of granulomatous reaction with well developed granulomas in lungs. Hyphae and conidiophores with fruiting bodies ofAspergillus fumigatus could be demonstrated in sections up to 24 days of infection. Reisolation of the fungus consistently was achieved up to 24 days. It is concluded that intratracheal inoculation ofAspergillus fumigatus spores in goats leads to pulmonary aspergillosis up to 24 days.     

Biological control of apple blue mold with Pseudomonas fluorescens

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.     

Destruction and formation of toxins by one bacterial species affect biodegradation by a second species

Two strains of Pseudomonas able to grow on phenol or p-nitrophenol (PNP) were isolated from sewage. Pseudomonas sp. PN101 mineralized and formed nitrite from PNP but did not mineralize phenol, and Pseudomonas sp. PH111 mineralized phenol but not PNP. Phenol increased the lag period before Pseudomonas sp. PN101 grew on and mineralized PNP, but this toxicity was reduced by inoculation of the medium with Pseudomonas sp. PH111. PNP inhibited growth of Pseudomonas sp. PH111 and slightly increased the length of the acclimation period for the mineralization of phenol by the bacterium. Inoculation of Pseudomonas sp. PN101 into solutions containing PNP and phenol increased the lag period prior to growth of Pseudomonas sp. PH111 on phenol and markedly lengthened the lag period for its mineralization of phenol. Coinciding with this delay in the onset of phenol degradation was the accumulation of an organic compound formed from PNP by Pseudomonas sp. PN101. This compound was not mineralized by the phenol-degrading bacterium. The data suggest that bacteria may interact during the decomposition of chemical mixtures by destroying or by forming toxins that affect the biodegradation of individual components of those mixtures.     

Cross Protection Between Strains of Fusarium oxysporum f. sp. vasinfectum and its Effect on Vascular Resistance Mechanisms

The severity of symptoms induced in cotton plants by stem inoculation with Fusarium oxysporum f. sp. vasinfectum was reduced by inoculation with a non-pathogenic strain (NPS) of the fungus 3 days previously. Symptom reduction was associated with reduced populations of the pathogen in the stele tissue of the stem. This cross-protective response became effective within 24 h of inoculation with the NPS. Although both vascular occlusion and terpenoid aldehyde synthesis were induced by theNPS, the short time required for cross protection to become effective and the duration of the effect, indicated that vascular occlusion rather than fungitoxicity of the terpenoid aldehyde compounds was the mechanism of protection.     

Bacterial growth in ground beef prepared from electrically stimulated and nonstimulated muscles.

Ground beef samples prepared from electrically stimulated and nonstimulated biceps femoris and infraspinatus muscles were inoculated with Lactobacillus sp., Pseudomonas sp., Acinetobacter sp., or a mixture of Lactobacillus spp., Pseudomonas spp., Acinetobacter spp., Moraxella sp., Microbacterium thermosphactum, and Erwinia herbicola. There were no significant differences in growth of various bacteria in ground beef made from electrically stimulated and nonstimulated muscles.     

Quantitative and qualitative studies of the microflora of barley malt production

Populations of aerobic heterotrophic bacteria, mycelial fungi and yeasts occurring in malting barley were estimated by a plate technique and scanning electron microscopy. There was an increase in the total number of micro-organisms during germination, although populations declined after kilning. Bacteria dominated on all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious spatial distribution of micro-organisms on the samples although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of the kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicum, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filamentous fungi were identified as Aiternaria alternata, Aspergillus glaucus (group), Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, C. vini, Debaryomyces hansenii, Hansenula polymorpha, Kloeckera apiculata, Rhodotorula mucilaginosa, Sporobolomyces roseus and Trichosporon beigelii.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.