In Norway, early application of fungicides against cereal leaf diseases (before Zadoks 60) is common practice amongst farmers. Whether this procedure has any effect on Fusarium infection of the mature grain has been little investigated. To evaluate effects on Fusarium grain infection, cereal grains were sampled during 1996, 1997 and 1998 from 12 field trials where early spraying against fungal diseases in spring wheat, spring barley and oats was carried out. Percentage infected grains and frequency of different Fusarium species was analysed in every grain sample. The effect of fungicides, glyphosate and postemergence herbicides on Fusarium grain infection was studied. Significant increase in Fusarium infection was detected in fungicide-treated plots compared with untreated plots. Fusarium avenaceum and F. tricinctum were the most frequent species detected. The internal ranking of Fusarium species remained the same after spraying. No significant effects were found on the level of Fusarium infection after glyphosate treatment in autumn or herbicide treatment during the growing season.
Yeast exo-β-1,3-glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 103 to 80 × 103 spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 103 to 80 × 103, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5-day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β-glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea, respectively, could be considered.
Very little is yet known regarding the molecular mechanisms involved in pathogen defense responses in citrus fruit. Recently, a basic β-1,3-endoglucanase (EC 184.108.40.206) belonging to the pathogenesis-related (PR) group of proteins, has been purified from Citrus sinensis (L) Osbeck cv. `Valencia' orange callus. Specific antibodies raised against the purified protein were used to screen `Valencia' callus and flavedo cDNA expression libraries, and to isolate its corresponding cDNA, designated gns1. The gns1 gene encodes a predicted polypeptide of 336 amino acids with a molecular mass of 37.3 kDa and a basic pI of 9.19, and shares 55–65% identity with several other plant β-1,3-endoglucanase proteins. Hereby, we show that the expression of the gns1 gene is markedly induced by wounding and inoculation with Penicillium digitatum (Pers. Fr.) Sacc., and following treatments with various elicitors that induce fruit resistance against P. digitatum. These treatments include UV irradiation, application of jasmonic acid (JA), β-aminobutyric acid (BABA), Candida oleophila antagonist yeast cells and hot water rinsing and brushing. Overall, based on various RNA gel blot hybridizations, we assume that gns1 is most likely to be part of the molecular mechanisms involved in pathogen defense responses in citrus fruit. †
Inoculation of different bean cultivars with Colletotrichum lindemuthianum race β results in a marked increase of β-1,3-glucanase and chitinase activities. The increase is much faster in incompatible than in compatible interactions. Induced β-1,3-glucanase (pI 9,5) differs from the constitutive β-1,3-glucanase (pI 4,5) of healthy plants. The induced enzyme can partly degrade, in vitro, the cell walls of C. lindemutianum. The possible role of these hydrolytic enzymes inplants defence is discussed.
A β-1,3-glucanase (GLU-39) was isolated from a potato cultivar with a high level of field resistance (Solanum tuberosum L. cv Huinkul), after 72 h of infection with Phytophthora infestans when β-1,3-glucanase activity was markedly increased. Purification was performed by anion exchange chromatography, gel filtration and by chromatofocusing. A size of 39 kDa was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in denaturing conditions and by gel filtration, indicating the monomeric nature of GLU-39. The enzyme produces a direct inhibitory effect on the germination of sporangia of P. infestans. Differential expression (determined by immunoblotting assays) of GLU-39 was observed between two potato cultivars with different degrees of field resistance to P. infestans. In the resistant cultivar (cv Pampeana INTA) GLU-39 induction (four-fold with respect to healthy tubers) occurred 14 h after inoculation and remained over basal levels at 38 h after inoculation. By contrast, in the susceptible cultivar (cv Bintje), GLU-39 was induced at lower levels than those observed in cv Pampeana INTA, and no differences were detected between wounding and infection. The localization, expression pattern and in vitro activity suggest that GLU-39 may have a major role in field resistance.
The virulent strain Ds 1 of Xanthomonas campestris pv. vesicatoria multiplied in pepper (cv. Hanbyul) leaves better than did the avirulent strain 81–23, which formed localized necrosis at the onset of pathogenesis. Infection of pepper leaves by X. campestris pv. vesicatoria induced the synthesis and accumulation of β-1,3-glucanase and chitinase in the intercellular space and leaf tissue of pepper plants. In the uninoculated controls, the two hydrolases remained at a very low level. High levels of the two enzymes were found in an incompatible interaction of pepper leaves with X. campestris pv. vesicatoria. In particular, chitinase activity in the intercellular washing fluids (IWF) was higher in the incompatible than in the compatible interactions. The direct detection of acidic β-1,3-glucanases on 10% native PAGE gels revealed only two isoform bands (Ga 1 and Ga 2). Isoelectric focusing identified two acidic β-1,3-glucanase isoforms with pl 5.0 and 5.2, and four basic isoforms with pl 7.1, 7.4, 7.9, and 8.8 in the IWF and extracts of infected leaf tissues. Some of the isoforms disappeared during pathogenesis and the others appeared during symptom expression. The acidic chitinase isoforms (Ca 1, Ca 2, and Ca 3) were located primarily in the intercellular spaces. Synthesis of high levels of the acidic isoform Ca 3 in infected pepper leaves was seen. Several basis chitinase isoforms accumulated only in diseased leaf tissue, and especially more in the incompatible than the compatible interaction. By using isoelectric focusing, the three acidic and seven basic chitinase isoforms in the IWF and leaf extracts were detected on chitin overlay gels.
β-1,3-glucanase and chitinase activities were induced locally and systemically 4–25 and 11–25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14–18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18–21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemicallyprotected unchallenged coffee leaves.
Following the addition of 0.2 mg 2,4-dioxohexahydro-1,3,5-triazine (DHT, formulation CKB 1130) to 1 l Gamborg's medium for organogenesis, the two viruses strawberry mottle virus and strawberry crinkle virus could not be detected in 74.4, 63.6 or 65.1%, respectively, of the plants regenerated from 0.8mm apices of the varieties Redgauntlet, Vesper and Pokahontas after their inoculation onto the indicator strain of Fragaria vesca EMK. In contrast, control explants which were not exposed to DHT proved to be 100% virus infected.
Suitability of in vitro potato plantlets for testing of antiphytoviral effect of a combination of 2,4-dioxo-hexahydro-1,3,5-triazine (DHT) and N-cyano guanidine
The suitability of a semiquantitative determination of virus content for the detection of antiphytoviral effects of chemicals was demonstrated by ELISA directly applying at in vitro potato cultures systemically infected and chemotherapeutically treated. The combination of 2, 4-dioxohexahydro-1,3,5-triazine (DHT) and N-cyanoguanidine both applied at a concentration of 0.03% to the culture medium resulted in a high significant reduction of the relative concentration of the potato virus S in the, potato genotype ‘M-812820’ of 44–73 %. A phytotoxic influence of the substances was not observed. The combination of the two antiphytoviral substances had no effect against potato virus Y in explants of the variety ‘Ackersegen’. Differences in the susc, eptibility of various genotypes against biologically active substances were indicated.
Die Eignung des semiquantitativen Virusnachweises mittels ELISA direkt an chemotherapeutisch behandelten systemisch infizierten In-vitro-Kulturen der Kartoffel zum Nachweis antiphytoviraler Wirkungen wurde untersucht. Die Kombination von 2,4-Dioxohexahydro-l,3,5-triazin (DHT) und N-Cyanoguanidin ergab bei Konzentrationen von je 0,03% zum Nährmedium eine hoch signifikante Senkung der relativen Konzemration des Potato vims S im Kartoffelgenotyp ‘M-8-12820 um 44—73%, ohne daß die Explantate Schäden erkennen ließen. Eine Wirkung des Kombinations-präparates gegen Potato virus Y in Explantaten der Sone ‘Ackersegen’ konnte nicht nachgewiesen werden. Sortenunterschiede in der Empfindlichkeit gegenüber biologisch aktiven Verbindungen deuten sich an.
Antiphytoviral activities of 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine
Substitution of 2,4-dioxohexahydro-1,3,5-triazine (DHT) by two acetyl groups resulted in an antiphytoviral compound, 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DA-DHT), which inhibits PVX, PVY, ToMV, TMV and TRV better than DHT, but only after, application at relatively high concentrations (10−2 mol/1). The antiphytoviral activity of DA-DHT is enhanced by combined treatments with DHT as well as with cyanoguanidine, sodiumalkanemonosulfonate and 2-anilino-5-adamantyl-1,3,4-thiadiazole., DA-DHT reduced the number of symptoms of PLRV bearing potato eye cutting plants to a higher percentage than DHT. But the highest reduction was brought about by combined treatments with DA-DHT, cyanoguanidine, sodiumalkanemonosulfonate and 2-anilino-5-adamantyl-1,3,5-thiadiazole. Moreover, treatments with DA-DHT increased more than treatments with DHT the mass of potato tubers produced by potato eye cutting plants. Combined treatments with DA-DHT and the above mentioned substances reduced the natural infection with PLRV of a completely healthy potato stock for about 70 % and that of PVY for about 40 %. Thus, the DA-DHT containing preparations may be capable of keeping potatoes in a good state of health even in regions with a high infection pressure.
Substitution von 2,4-Dioxohexahydro-l,3,5-triazin (DHT) durch zwei Acetylgruppen führte zu einer antiphytoviralen Verbindung, dem l,5-Diacetyl-2,4-dioxohexahydro-l,3,5-triazin (DADHT), die PVX, PVY, ToMV, TMV und TRV besser als DHT hemmt. Allerdings ist hierfür die Applikation relativ hoher Wirkstoffkonzentrationen, z. B. 10-2 mol/1, erforderlich. DA-DHT verminderts auch die Zahl von Augensteckiingspflanzen der Kartoffel mit Symptomen des PLEV stärker als DHT. Die an entsprechenden Pfknzen gebildete SLnolienmasse wurde durch DA-DHT in größerem Maß als durch DHT erhöht. Die antiphytovirale Wirkung des DA-DHT wird beträchtlich verstärkt, wenn diese Substanz in Kombination mit DHT oder Cyanoguanidin, Natriumalkanmonosulfat bzw. 2-Aiulmo-5-adamantyl-l,3,4-thiadiazol appliziert wird. Eine kombinierte Behandlung mit den genannten vier Verbindungen verminderte die Zahl der Augenstecklingspflanzen mit Symptomen des PLRV besonders stark. Ebenso verminderte mehrfache Behandltmg mit der angeführten Substanzkombination die natürliche Infektion einer gesunden Kartoffelherkunft mit PLRV um etwa 70 %, die Infektion von PVY um 40 %. Damit könnten DA-DHT-haitige antiphytovirale Präparate dazu beitragen, den Virusbesatz von Kartoffelherkiinften selbst in Gebieten mit hohem Infektionsdruck niedrig zu halten.
In two consecutive trials, three treatments of tomato plants of the variety Rivermoon with the antiphytoviral substance DHT (2,4-dioxohexahydro-1,3,5-triazine, 0.15%) 2 d a.i. and 2 and 7 d p.i. reduced the concentration of ToMV (tomato mosaic virus) by 62.3 and 60.5%. The average tomato yields increased by 40 and 26 % compared with the virus diseased, untreated controls, but the yields of the healthy controls were not achieved. A combined treatment of DHT, alkane monosulphonate and N-phenyl-N'-p-carboxyphenyl thiourea only resulted in a minor reduction in the ToMV titre and only a slight increase in yield, compared with the DHT-solo treatment.
Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1,3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants.
Alternaria alternata, is the predominant fungal pathogen responsible for moldy-core in apple cultivars of the Red Delicious group. Here we report on the association between virulence of natural isolates of A. alternata, their production of endo-1,4-β-glucanase (EG) and moldy-core development in apple fruits. Based on decay development following wound inoculations of mature fruits, three of 150 isolates, collected in three orchards in northern Israel and representing low, moderate and high virulence, were selected and designated Rm44, Er30 and Sh42, respectively. All three isolates secreted EG when grown on enzyme-inducing medium (EIM) containing commercial cellulose or apple cell walls and this production was related to their degree of virulence. Polyacrylamide gel electrophoresis (PAGE) revealed quantitative differences between the three isolates, relative to their virulence. When fungal extracts were run in native gels, a single band with a molecular mass of 23 kDa showing EG activity was produced by the high- (Sh42) and the medium-virulence (Er30) isolate but not by the low-virulence (Rm44) isolate. A commercial cellulase preparation (containing endo- and exo-1,4-β-glucanase) placed on pricked fruit led to the formation of symptoms similar to those developing on A. alternata-inoculated fruits within 2–4 days. Inoculation of bloom clusters at full bloom with the highly virulent isolate (Sh42) of A. alternata resulted in a significantly higher infection in fruits (58%) than in those inoculated with the low-virulence isolate (Rm44) (30%). Our results suggest that the moldy-core symptoms caused by A. alternata in apple, could be related to the ability of the fungus to produce EG in developing lesions.
The study of the agent associated with the mild type of Hydrangea virescence in France involved three steps, with the aid of transmission electron microscope (TEM). In the first step, we observed the presence of polymorphic procaryotes in thephloem sieve tubes of diseased plants and their absence from corresponding healthy plant parts. The procaryotes were detected in the areas suspected in 1000 nm thick sections stained with thionin-acridine orange. In the next step, the ultrastructure of their unit membrane was studied at magnifications higher than 100 000. The two osmiophilic layers of the membrane were 6 nm distant and no preliminary parietal shape was detected. These observations on ultrathin (60 nm) sections allowed us to classify the, particles into the class “Mollicutes”. The third step involving the examination of 350–1000 nm thick sections revealed the absence of spiral forms.
The TEM observations are consistent with the hypothesis that the agents associated with the mild type of Hydrangea virescence observed in France should be included within the MLO group. A method specially adjusted to the fixation of MLO inside sieve tubes has been mentioned.
The slow growth of many filamentous fungi in vitro prevents rapid isolation of large quantities of genomic DNA, thereby hampering molecular studies. Using a polymerase chain reaction (PCR)-based adapter ligation cloning technique that requires only nanogram quantities of genomic DNA we have successfully cloned the complete CYP51 gene encoding the eburicol 14α-demethylase, the target for DMI (Demethylation-inhibiting) fungicides, from the Japanese pear scab fungus, Venturia nashicola. Analysis of predictedamino acid sequences of CYP51s from strains less sensitive to DMIs revealed no alterations when compared to the sensitive reference strain.
Time course absorption and desorption of metalaxyl by seeds of pearl millet was analysed by following chemical kinetics equations. Uptake of metalaxyl through roots, leaves and seed, its translocation and distribution in different plant parts and persistence following seed application were studied in pearl millet using 14C-metalaxyl. Both uptake and efflux of metalaxyl by pearl millet seeds were complex and compartmentalized. Distribution inside the seed was not uniform. A major part of applied fungicide remained within the treated plant part, particularly after seed and foliar applications. Metalaxyl was ambimobile inside the plant and was found to get accumulated at apex and margins of leaf blade. No metalaxyl could be detected in grains, harvested from plants grown from metalaxyl treated seeds.
Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (FST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1-5.8S-ITS2) restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis. PCR-RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.
Ralstonia solanacearum is a β-proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain-specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex.
The lipopolysaccharide (LPS) side chain from Pseudomonas syringae pv. tabaci strain NCPPB 79 (=CFBP 1615) contained L- and D-rhamnose, and GlcNAc. Using methylation analysis, periodate oxidation, Smith degradation and H-1- and C-13-nuclear magnetic resonance spectroscopy, the repeat unit was found to have the structure: 3)-alpha-D-Rhap(1 -> 3)-beta-L-Rhap(1 -> 4)-alpha-L-Rhap(1 -> 4 up arrow beta-GlcNac(1 This structure is correlated with a previously proposed serogrouping system. The involvement of LPS generally in plant disease is briefly discussed.
Two phytoplasmas closely related to the X-disease group were associated with China-tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China-tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China-tree and garlic decline phytoplasmas to the 16SrIII (X-disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China-tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X-disease group.
A rapid polymerase chain reaction (PCR)-based procedure was developed for the detection of Pseudomonas avellanae, the causal agent of hazelnut (Corylus avellana) decline in northern Greece and central Italy. The partial sequence of the 16S rRNA gene of P. avellanae strain PD 2390, isolated in central Italy, was compared with the sequence coding for the same gene of P. syringae pv. syringae type-strain LMG 1247t1. Primers PAV 1 and PAV 22 were chosen, and after the PCR, an amplification product of 762 base pairs was specifically produced only by 40 strains of P. avellanae isolated from northern Greece and central Italy. No other bacterial species among those tested showed an amplification product under optimized PCR conditions. The adding of 4% BLOTTO (10% skim milk powder and 0.2% NaN3) in the PCR mixture proved essential in order to avoid interference of hazelnut extracts during the amplification. The procedure proved more effective than repetitive PCR with ERIC primer sets in diagnosing apparently healthy hazelnut trees as infected. This technique could be of great help for screening the hazelnut propagative material as well as for monitoring the wild C. avellana trees growing in the woods near the infected hazelnut orchards.
The genetic relationship between faba bean (Vicia faba L.) phyllody and other mycoplasma-like organism (MLO) diseases has been studied by amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using Alu I restriction endonuclease. The restriction patterns produced by faba bean phyllody MLO were smilar to that of Crotalaria saltiana phyllody MLO which persists throughout the year in the Sudan. These, and serological results clearly confirmed that C. saltiana is a reservoir of faba bean phyllody MLO in the Sudan. Moreover, restriction patterns have also shown that MLOs of other diseases have the same RFLP fragment pattern as faba bean phyllody MLO, including C. juncea witches'broom (Thailand) and tomato big-bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, aqll from France).
Fragment patterns also revealed the existence of genetically diverse MLO strains in the Sudan. Faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flaveswcence dorée of grapevine, plum leptonecrosis of Prunus salciana, peachy yellow leaf roll, sunnhemp phyllody from Thailand, and blueberry witches' broom.
A new form of disease called ‘die-back’ has been established in Dalbergia sissoo trees. This disease has reached epidemic proportions in Bangladesh as well as in other countries of South Asia and is characterized by browning of the leaves, signs of wilting, and trunk lesions with gum flow. The trees die within a few months. In order to investigate the causes of this die-back disease, samples were taken for a first trial in the Rajshahi division at two sites around Sherpur. For the isolation of bacteria, surface-sterilized plant material (leaves, twigs and trunk bark) from diseased trees was transferred to LB medium and incubated. After isolation of single colonies, various bacteria species could be identified by polymerase chain reaction analysis with two primers specific for highly conserved sequence regions in the bacterial 16S rDNA and by sequencing. First indications for the presence of bacteria with phytopathogenic potential were found.
Stylosanthes sp. exhibiting characteristic symptoms such as little leaf, witches’ broom and floral abnormalities were collected from north Queensland and the Northern Territory, Australia. Previous studies have shown that sweet potato little leaf V4 (SPLL-V4), tomato big bud (TBB), stylosanthes little leaf (StLL) and pigeon pea little leaf (PLL) phytoplasmas are associated with this disease. The detection of an additional phytoplasma type, vigna little leaf (ViLL) is reported herein. The range and severity of symptoms expressed by affected plants is highly variable and is not associated with a particular phytoplasma type. Similarly, host plants infected with a complex of two phytoplasmas did not have unique or more severe symptoms. Of the phytoplasmas associated with stylosanthes little leaf disease, StLL is unique because it lacks the tRNAIle gene which is normally situated in the 16S-23S rRNA intergenic spacer region. This phytoplasma was shown to have a second operon containing the expected tRNAIle gene in all StLL samples examined. Sequence analysis suggests that the two 16S rRNA genes amplified by polymerase chain reaction from StLL samples originate from the same phytoplasma. This the first report of a phytoplasma having ribosomal operons both with and without an intergenic tRNAIle gene.
Phytoplasmas are associated with several plant diseases occurring in Brazil. A phytoplasma of group 16SrIII found in tomato plants with symptoms of big bud was identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA. RFLP patterns using HhaI and RsaI endonucleases were distinct from those exhibited by phytoplasmas representatives of diverse subgroups of group 16SrIII. Nucleotide sequence analyses demonstrated sequence heterogeneity expressed through a few base positions and restriction site among cloned fragments, revealing lineages different from members of currently known subgroups. The detection of lineages within tomato big bud phytoplasma present in Brazil revealed the diversity of representatives of group 16SrIII in tropical ecosystem and confirmed the genetic diversity of phytoplasmas of that group around the world.
Nearby fruit and vegetable fields in Islamabad, Pakistan were surveyed for phytoplasma infection. ‘Candidatus Phytoplasma asteris’ (Group 16SrI) was found infecting mango, citrus, loquat, geranium, periwinkle, radish, blackberry and potato. Results suggest that a polyphagous vector may be involved in phytoplasma transmission to these plant species, which are first host records of 16SrI phytoplasma infection in Pakistan.
Yellow leaf syndrome was a serious problem in the beginning of the 1990s in Brazil, when yield losses were estimated to be around 50%. The disease is currently endemic, but it is considered potentially important. Previous studies have revealed only the presence of a luteovirus associated with the disease in Brazil. We report that a phytoplasma of 16SrI-B is also associated with this disease. This is the first demonstration of the presence of a group 16SrI-B phytoplasma in association with sugarcane yellow leaf in Brazil.
Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea, the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 106, 1 × 107, 1 × 108) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.
Recently, a epidemic of apple proliferation (AP) in an orchard area of Trentino (North Italy) occurred. The most affected cultivars were Golden Delicious, Florina and Renetta Canada grafted on different rootstocks. In this area the known or supposed vectors of the disease were not present. At the same time as the AP symptoms appeared, a notable increase of the presence of psyllids was observed on apple trees so a correlation between these insects and the AP was hypothesized. Four different psyllid species were found in the orchard: Cacopsylla melanoneura (Forster 1848), Cacopsylla costalis (Flor 1861), Cacopsylla mall (Schmidberber 1836) and Trioza urticae (Linnaeus 1758). The first two were more frequent in spring at the adult stage. In 1997 C. costalis was particularly numerous and was used to plan AP transmission trials. The transmission from infected AP apple trees to the insect in field and from the psyllids to non-infected Golden Delicious and Florina plants in the greenhouse were conducted. For the control of phytoplasma presence in the psyllids and in the apple plants polymerase chain reaction (PGR), nested PCR and restriction fragment length polymorphism analysis were used. In autumn 1997, some inoculated plants showed symptoms of AP. Molecular results were consistent with the presence of phytoplasma in C. costalis and in inoculated apple cultivars.
Karl Otto Müller was an outstanding scientist in applied botany who substantially promoted modern plant breeding and phytopathology. In 1928 he started his experimental studies about phytophthora resistance of potatoes enlightening on an up to that time unknown defensive reaction of higher plants, which led to the postulation of phytoalexins 50 years ago.
Thirty-four isolates of Verticillium dahliae Kleb. from nine different genera of dicotyledonous host plants and a broad range of geographic regions were analysed genotypically, Random amplified polymorphic DNA (RAPD) markers were used for the estimation of the genetic variability within the species. Using four primers for the analysis, 79 distinct fragments were obtained. The derived phenogram clustered the isolates in two main groups; one consisted almost entirely of V. dahliae isolates from oilseed rape (Brassica napus napus), the other group comprised isolates from a wide range of host plants. No correlation between geographic location of the isolates and the RAPD-pattern was observed.Sequencing of the gene for the 18SrRNA and calculation of the phylogenetic tree integrated the deuteromycetous fungus V. dahliae into the sexual system of the filamentous ascomycetes.
Among the bacterial strains isolated from diseased sunflower leaves, eight were studied in some detail. A fluorescent pseudomonad isolated from necrotic tissues and its reisolates belong to group Ia of phytopathogenic pseudomonads which includes Pseudomonas syringae bacterium. A study of host range indicated that the pathogen infects only sunflower but not the other plant species. Based on the pathogenicity study and biochemical and physiological tests, it was concluded that the pathogen belongs to the bacterium Pseudomonas syringae pv. helianthi.
Pseudomonas syringae pv. helianthi (Kawamura 1934) Dye, Wilkie et Young 1978, ein Pathogen der Sonnenblume
Von einigen Bakterienstämmen, die von Sonnenblumenblättern isoliert worden waren, wurden acht Stämme näher untersucht. Ein fluoreszierender Pseudomonade, aus nekrotischem Gewebe isoliert, sowie dessen Reisolate gehören der Gruppe la der phytopathogenen Pseudomonaden an, Pseudomonas syringae gehört auch zu dieser Gruppe. Eine Untersuchung des Wirtspflanzenspektrums des Pathogens zeigte, daß das Bakterium nur Sonnenblumen und keine andere Pflanzenart infiziert. Basierend auf den Pathogenitätsuntersuchungen sowie biochemischen und physiologischen Studien wurde das Bakterium als Pseudomonas syringae pv. helianthi bestimmt.
Determinative laboratory tests which distinguish Rhodococcus fasdans from all other coryneform plant pathogenic bacteria were used to compare strains of this species with representative strains of Clavibacter spp. and Curtobacterium spp. The strains were also compared by colony hybridization using genomic DNA from R. fascians as a probe. Only strains of R. fascians authenticated with the determinative tests gave positive reactions to the probe.
The pathogenicity of strains was tested in seedlings of Lathyrus odoratus L. (sweetpea) grown in soil, and asepticaily in agar in tubes. Fasciation was induced in sweetpea by 20 strains of R. fascisms from Begonia sp., Brodiaea laxa, Carica X heilbornii nm. pentagona, C. pubescens, Dahlia sp., Fragaria X ananassa, Lathyrus odoratus, and Verbascum nigrum. Strains of R. fasdans from Chrysanthemum sp., Gladiolus sp., and Justicia brandegeana did not induce fasciation in sweetpea.
This study was conducted to access the ability of fluorescent Pseudomonas spp. to suppress cyst nematode Heterodera cruciferae, which was caused by reproduction. The yield loss of cruciferous plants in infested soil with H. cruciferae was examined in in vitro and in vivo experiments. For this purpose, fluorescent Pseudomonas spp. was isolated from infested soil with cyst nematode H. cruciferae and a selected strain of fluorescent Pseudomonas [Fluorescent Pseudomonas strain no. 6 (FPs6)] used on cyst, females and eggs of H. cruciferae to study the biological control. In the in vitro study, eggs of H. cruciferae were infected by FPs6 on King's B Agar and consequently the growth and hatching of eggs were inhibited. Furthermore, J1 and J2 being grown in diseased eggs as they were infected by FPs6. In the in vivo study, plant lengths, leaf areas, wet weights, dry weights and root lengths of seedlings were investigated. There were no considerable differences among FPs6, FsP6 + Cyst and Control groups on plant length and root length (P > 0.05). But there were considerable differences between Cyst and Control groups on plant length and root length (P > 0.05).
During 1985 60% of Claviceps purpurea sclerotia collected from Triticale heads were colonized by Fusarium acuminatum Ell. et Ev. and F. heterosporum Nees.
Besiedlung von Sklerotien von Claviceps purpurea auf Triticale durch Fusarien
60% der Sklerotien von Claviceps purpurea auf Ähren von Triticale waren 1985 durch Fusarium acuminatum und F. heterosporum befallen.
Representative random samples of spores of Erysiphe graminis f. sp. hordei, causing powdery mildew of barley, were taken from the atmosphere by means of a Schwarzbach jet spore sampler over important barley growing areas in Europe and tested in the laboratory for their virulence on host plants with resistance Ml 41/145, Mlg, Mla6, Mla12, Mla7+Mlk, MlLa, Mla1, Mla9, Mla3, Mla13+Ml (Ru3) and mlo. Virulence frequencies mostly showed regional differentiation and efficiency of, the resistance factors generally increased in the order given. Frequency and level of resistance against triadimenol also showed significant regional differences. Against fenpropimorph similar levels of sensitivity were observed in different parts ofEurope, close to that of standard-isolates, which had a mean position. The results are discussed with respect to current and previous conditions of cultivation, to the spread of the pathogen by wind and towards strategies for more effective use of hostresistance and of fungicides.
Isolates of Phytophthora infestans were collected from all potato growing regions of Poland during the blight seasons of 1987—1989. All 1987 isolates were of Al mating type and were sensitive to metalaxyl. In 1988 and 1989, 46.5 % and 55.3 % of the isolates were sensitive to metalaxyl, respectively. The percentage of highly resistant (R) isolates increased from 25.6 % in 1988 to 39.5 % in 1989; however the percentage of intermediately resistant (I) isolates decreased during that period from 27.9 % to 5.3 %. A significant association was observed between the A1 compatibility type and metalaxyl resistance. The A2 mating type first appeared in 1988, and its frequency increased from 4.7 % of the population in 1988 to 47.6 % in 1989. Coincident with this change in mating type frequency, changes in ploidy levels of isolates were observed. Whereas 3 % of the 1988 isolates were diploid, 90 % of the 1989 A2 isolates and 28.6 % of the 1989 Al isolates were diploid. The approximate 1:1 ratio of the two mating types encountered in 1989, and the predominance of diploidy, indicates that the Polish population of P. infestans has the potential to become sexual.
A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Strr) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Strr strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.
Die Verbreitung von Streptomycin-resistenten Stämmen von Erwinia amylovora in Ägypten im Jahre 1988
Während der Monate April und Mai 1988 wurde in einigen ägyptischen Birnenplantagen eine Studie durchgeführt, um das Auftreten von Erwinia amylovora-Stämmen zu ermitteln, die gegen Streptomycin resistent sind. 22 aus insgesamt 604 Isolaten aus 11 Plantagen zeigten eine Resistenz gegen Streptomycin, wobei diese alle gegen hohe Streptomycinkonzentrationen immun waren. Dabei lagen die minimalen Hemmungskonzentrationen zwischen 1000 und 3000 μg/ml. Das Vorkommen Streptomycin-resistenter Stämme von Erwinia amylovora ist in Ägypten noch begrenzt, und die Populationen dieser Stämme sind auch nicht sehr groß. Dennoch ist das Auftreten dieser resistenten E. amylovora-Stämme eine potentiell gefährliche Situation.
Samples of grain, harvested in October/November of 1993 or in the spring of 1994 from fields in Norway with overwintered grain, were collected. The presence of Fusarium species and the amount of mycotoxins produced by this genus were determined. The cytotoxic properties of the grain samples were examined with an in vitro methylthiazoltetrazolium (MTT)-cell culture assay using swine kidney and VERO cells as target cells. The total count of colony forming units of Fusarium species was about the same in the grain harvested in late autumn and in the spring, but the dominant species seemed to vary somewhat between the two groups. F. culmorum was found more in the samples from October/November while F. avenaceum was isolated more in the grain harvested in the spring. Only small amounts of F. sporotrichioides were present in the grain harvested in the autumn, and none was found in the overwintered grain. The deoxynivalenol content was significantly higher in the grain harvested in autumn than in the overwintered grain. Although very small amounts of toxins were detected in the overwintered grain, it was more cytotoxic than the grain harvested in October/November. A significant correlation between the cytotoxicity and the amount of F. avenaceum in the samples was found.
The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty-five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.
A total of 241 isolates of Phytophthora infestans were collected in 1997, 2006 and 2007 in eight European countries and characterized with molecular markers (simple sequence repeats, SSR genotypes) and phenotypic traits such as sensitivity to fungicides, mating type and aggressiveness. The mating type distribution changed from mainly A1 in 1997 to a majority of A2 in 2007. No resistant isolates were detected for fluazinam and mandipropamid, whereas the proportion of isolates resistant to mefenoxam (MFX) was high and increased over the years. There was no genetic link between mating type and MFX resistance. Aggressiveness (product between lesion expansion and sporulation capacity) was slightly higher for MFX-resistant compared to sensitive isolates and for isolates collected later compared to earlier in the same season. It was about equally high for A1 and A2 types, and for French isolates in 1997 and British isolates in 2007, but lower for French isolates in 2007. Six different SSR genotype families were distinguished. In 1997, populations were dominated by genotype families I and III/IV, which significantly declined in 2007 being largely displaced by genotype families II (‘blue 13’ type) and V, which are by coincidence mainly A2 MFX resistant and A1 MFX sensitive, respectively. However, mating type and MFX resistance were genetically not linked to SSR genotypes.
Wheat leaf rust (Puccinia triticina) is becoming a serious concern in Spanish wheat, especially on durum wheat where acreage has enormously increased. Host resistance is the preferred method of disease control, but the virulence spectrum of the leaf rust population in Spain is currently unknown. In order to deploy effective Lr genes, this study was conducted to characterize the virulence spectrum of leaf rust in Andalusia (Spain). Isolates were obtained from surveys of wheat fields across Andalusia from 1998 to 2000. From 56 isolates phenotyped, 35 pathotypes were identified. Virulence to Lr10, Lr11, Lr14a, Lr14b and Lr18 was high (>96%), while virulence to Lr9 and Lr24 were not found. None of the isolates collected from durum wheat were virulent to Lr1, Lr3, Lr3ka, Lr3bg, Lr15, Lr16 and Lr17, while many of the isolates collected on bread wheat showed virulence on these genes, indicating a certain specialization in the leaf rust infecting durum wheat. Population dynamics of current wheat leaf rust pathotypes in terms of mutation and migration are discussed.
In this study, an integrated approach was evaluated for the control of postharvest decays of mandarin including some pre- and post-harvest treatments under storage conditions. The efficacy of the treatments both as alone and in combination was evaluated during 3 years. Preharvest application of benomyl resulted in significantly less decay of mandarin fruit after storage in 3-year tests. Calcium chloride (CaCl2), 2,4-dichlorophenoxyacetic acid (2,4-D) and gibberellic acid (GA3) as stand-alone treatments or combinations were not effective in controlling Penicillium and total decay infections on inoculated mandarin. Postharvest application of imazalil (200 μg/ml) in solution heated to 54°C for controlling postharvest green and total decay of mandarin was significantly effective for 3 months under storage conditions. The biocontrol activity of yeast (Metschnikowia pulcherrima) was improved when yeast treatment was combined with imazalil (200 μg/ml) at postharvest. The data suggest that preharvest application of benomyl and postharvest treatments of imazalil, hot water and yeast may reduce postharvest green mould and total decay of mandarin under storage conditions.
The systemic acquired resistance (SAR) was studied in barley to find out specific molecular markers for this type of resistance. Such markers may serve as diagnostic tools to indicate the defense-status of a plant and, additionally, may be used to identify new resistance-inducing compounds in broad screening experiments. Upon treatment of barley leaves with the resistance-inducing compound 2,6-dichloroisomicotinic acid (INA) we found, in addition to a yet unidentified basic protein of 45 kDa (designated BIR-1) induction of a specific 6kDa polypeptide. Using Northern-and Western blot analysis this small polypeptide was identified as JIP-6, a known member of the jasmonate-induced protein (JIP)-family of barley that was previously demonstrated to be a leaf thionin (ANDRESEN et al. 1992).
Die systemisch erworbene Resistenz in der Gerste wurde mit dem Ziel untersucht, neue molekulare Marker zur Identifizierung des Resistenzstatus in der Pflanze und zum Auffinden neuer Resistenzinduktoren bereitzustellen.Wir berichten vom Auftcten eines 45 und cines 6 kDa Polypeptids in Pflanzen, die mit 2,6-Dichlorisonikotinsaure behandelt wurden. Mittels der Northern-und Wesrtern Blot Analyse wurde cin Polypeptid als das Jasmonat-induzicrnbare JIP-6 identfiziert, identisch mit einem Blatt-Thionin der Gerste ist.
The former phenotypic study of Erwinia amylovora (VANTOMME et al. 1982) was extended with a collection of 54 Erwinia amylovora strains from a broad plant and geographic origin. From the 85 phenotypic features studied, 72 (85%) were present in at least 90% of the strains. Only 49 (58%) of the features were shared by all strains. Thirty-eight strains were also examined by the API 20E system. The API 20E code numbers for E. amylovora are unique and, combined with an immature, (green) pear test, may be used for an accurate identification of Erwinia amylovora.
Charakterisierung weiterer Erwinia amylovora Stämme und die Anwendung des API 20E-Systems bei der Diagnose
Die frühere phänotypische Untersuchung von Erwinia amylovora (VANTOMME et al. 1982) wurde durch eine Sammlung von 54 Erwinia amylovora-Stämmen, die von einer Vielzahl von Pflanzen und einer breiten geographischen Herkunft stammten, erweitert. Von den untersuchten 85 phänotypischen Merkmalen waren 72 (85%) in mindestens 90% der Stamme vorhanden. Nur 49 (58%) der Merkmale waren bei alien Stämmen vorhanden. 38 Stamme wurden auch mit dem API 20E-System untersucht. Die API 20E-Codenummern für E. amylovora sind einmalig und konnen zusammen mit einem unreifen Bimentest für eine akurate Identifizierung von Erwinia amylovora benutzt werden.
Primary leaves of 10 barley cultivars were uniformly inoculated with 22 virulent powdery mildew isolates using a jet spore trap. The number of colonies per unit area leaf was counted. Significant differences in number of colonies among cultivars were found, indicating different degrees of susceptibility among cultivars. While cultivars were rather consistent in their rankings for number of colonies produced by most isolates, interactions between some cultivars and isolates were found. In spite of great differences in geographical origin of cultivars and isolates, the interactions were not associated with origin.
Dass. Gekürzte Fassg. Mit 6 Abb. S. 295-312. gr. 8 (Nur in beschr. Anz. f. d. Aust.) Aus: Phytopathologische Zeitschrift. Bd 44. 1962, H. 3 Göttingen, Landwirtsch. F., Diss. v. 26. Juli 1962 (Nicht f. d. Aust.).