The incorporation of pathogen spores into rain-splash droplets: a modelling approach

Simple, theoretical, physical principles and existing experimental data were used to derive an analytical model to describe the incorporation of plant pathogen spores into splash droplets. Data were obtained from experiments on splash dispersal of spores of Pseudocercosporella herpotrichoides (cereal eyespot), Pyrenopeziza brassicae (oilseed rape light leaf spot) and Septoria nodorum (wheat glume blotch). In these experiments, incident drops of diameter 4–5 mm were allowed to fall onto spore suspensions 0.5 mm deep with 1.2 × 10⁵ to 6.5 × 10⁵ spores/mL. The analytical model was constructed as the product of three functions of droplet diameter which described, respectively, the frequency distribution of droplet diameters, the proportion of droplets carrying spores and the mean number of spores in spore-carrying droplets in each diameter category. The frequency distribution of droplet sizes was described by a log-normal distribution, the proportion of droplets carrying spores was described by an exponential function and the adimensional spore concentration in spore-carrying droplets was described by a power law. The cumulative proportions of spores in droplets in diameter categories of increasing diameter were calculated to compare observed and fitted data.     

Pathogenicity and Virulence of the Two Dutch VCGs of Verticillium dahliae to Woody Ornamentals

Two experiments were performed in two consecutive years to test whether isolates of different vegetative compatibility groups (VCGs) differ in their ability to cause disease in woody ornamentals, to study the host specificity of the isolates and to get an insight into disease development in woody hosts. A range of woody ornamental plant species, including Acer campestre, Acer platanoides, Acer pseudoplatanus, Catalpa bignonioides, Cotinus coggygria, Robinia pseudoacacia, Rosa canina, Syringa vulgaris and Tilia cordata, were root-dip inoculated with six isolates of Verticillium dahliae, belonging to the two VCGs that occur in the Netherlands (VCG NL-1 and VCG NL-2). Isolates belonging to each VCG caused severe symptoms of verticillium wilt in most plant species tested. Disease progress differed between plant species, but was generally the same for the two VCGs. No overall differences in virulence were observed between the two VCGs for external wilt symptoms, number of dead plants, or shoot length. No significant VCG × plant species interactions were present for these characteristics. However, isolates of VCG NL-1 caused more vascular discolouration than did isolates of VCG NL-2. Isolates within VCGs often differed considerably in their virulence to certain hosts, as shown by highly significant isolate × plant species interactions. Isolates were more virulent on their original host. These findings imply that VCG identification does not contribute to disease prediction for a range of woody hosts.     

Characterization ofSolanum tuberosum simple sequence repeats and application to potato culiwar identification

With the continued introduction of new potato cultivars, accurate identification is becoming difficult but is essential for maintaining cultivar integrity and Plant Breeders’ Rights. Hypervariable DNA sequences, referred to as simple sequence repeats (SSRs) or microsatellites, have been reported to be an excellent source of genetic markers. To determine the abundance, distribution, and composition of SSRs withinSolanium tuberosum, 252 sequences were searched for tetranucleotide and smaller SSRs with a minimum length of 20 nucleotides and a maximum discrepancy of two nucleotides. In total, 40 unique SSRs were observed in the 252S. tuberosum sequences examined and occurred at a frequency of one SSR every 8.1 kb. To assess the ability of site-specific amplified SSRs to identify potato cultivars, a simple (TCAC)_m and compound (TCAC)_m ? (CTT)_n SSR 5’ to the starch synthase gene and a compound (C)_p ? (CT)_q ? (AT)_r ? (G)_s SSR 5’ to the sequence encoding mature proteinase inhibitor I, were examined and shown to produce unique DNA profiles for 73 of 95 tetraploid cultivars. In total, 24 alleles were observed at these loci and the accurately sized amplified DNA products can be used to establish a database for cultivar identification. Site-specific amplified alleles were somatically stable and have been conserved in clonal variants of Russet Burbank independently maintained for almost seven decades, a characteristic essential for cultivar identification. As genetic markers, the abundant, informative, and easily examined site-specific amplified alleles of SSRs are ideal for quickly and accurately determining cultivar identity of S.tuberosum ssp.tuberosum.     

Direct penetration of Rhizopus stolonifer into stone fruits causing rhizopus rot

Rhizopus rot, caused by Rhizopus stolonifer, is a major postharvest disease of stone fruits. The disease is related to the occurrence of mechanical and physical damage; however, observations at a Brazilian wholesale market suggest that direct penetration can occur. Therefore, the penetration mechanisms of R. stolonifer in stone fruits were evaluated. To identify the production of enzymes that help with direct penetration by the pathogen, esterase activity, both in mycelial discs and in spore suspensions of the fungus in water and in modified Van Etten nutrient solution, was measured. Assays were also conducted to evaluate the growth of R. stolonifer on glucose or cutin as a sole carbon source. The pathogen grew on both media, and higher esterase activity was observed in the cutin medium. Wounded and unwounded peaches and nectarines were inoculated with R. stolonifer spore suspensions in water or in modified Van Etten nutrient solution. Wounded fruit inoculated with either of the R. stolonifer spore suspensions developed rhizopus rot, whereas unwounded fruit developed the rot only in the presence of spores in the modified Van Etten nutrient solution. Scanning electron and light microscopic examination showed the fungus can directly penetrate the nectarine cuticle. Diisopropyl fluorophosphate, a serine hydrolase inhibitor, prevented rot development in peaches. The results provide valuable evidence for the ability of R. stolonifer to directly penetrate unwounded stone fruits, probably due to the production of esterase enzymes.     

Identification and characterization of bacteria isolated from crown galls on stone fruits in Poland

Eighty stone fruit nurseries located in different regions of Poland were examined for the presence of crown gall affected plants. The disease was observed in 39 nurseries, and galls were sampled for bacterial isolation. Out of 1213 isolates, 409 were pre‐identified as Agrobacterium/Rhizobium spp. with 23S rDNA‐based multiplex PCR, and out of these, 315 were pathogenic when tested on sunflowers. Sequence analysis of three housekeeping genes (fusA, recA, rpoD) revealed that 366 strains belonged to Rhizobium rhizogenes, 23 to Agrobacterium tumefaciens species complex, and the rest of the strains were allocated to new phylogenetic lineages. Of these, the most numerous was the lineage allocated in the Pararhizobium genus. Positive results obtained from pathogenicity tests were generally in agreement with results obtained by PCR with primers complementary to T‐DNA except for two strains, which were positive for PCR but negative for the pathogenicity test. All detected Ti plasmids were nopaline‐type. Independent of their pathogenicity, 59% of tested strains were not sensitive to agrocin 84 in in vitro tests. Analysis of biochemical and physiological features distinguished 50 groups with different phenotypic profiles, but the tested traits were not consistent for strains classified to one taxon. This finding shows limited value of biochemical tests in identification procedures. The bacteria causing tumours were heterogeneous and strains classified to different taxa were found even in a single tumour.     

Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.     

Wound contamination and antimicrobial susceptibility of bacteria cultured during total ear canal ablation and lateral bulla osteotomy in dogs.

OBJECTIVE: To detect contamination of wound sites from surgical handling of excised tissues during total ear canal ablation and lateral bulla osteotomy in dogs, and to compare susceptibility of bacterial isolates to cefazolin with susceptibility to other antimicrobial agents. DESIGN: Prospective clinical study. ANIMALS: 13 dogs. PROCEDURE: Dogs were treated surgically for otitis externa and media via total ear canal ablation and lateral bulla osteotomy. Specimens for aerobic bacterial culture were obtained from SC tissue immediately following skin incision, tissues excised from the osseous bulla (after transection of the horizontal ear canal and lateral bulla osteotomy), and from SC tissue prior to skin closure. Antimicrobial susceptibility of bacterial isolates to various antibiotics was determined by use of a broth dilution assay. RESULTS: There was a significant association between isolation of Streptococcus canis and Escherichia coli from specimens from the osseous bulla and specimens from the SC tissues prior to skin closure, indicating contamination of the SC tissues during surgery. Seventy percent of bacterial isolates were susceptible to cefazolin. CLINICAL IMPLICATIONS: Measures to limit bacterial contamination resulting from tissue handling during total ear canal ablation and lateral bulla osteotomy are necessary. Bacteriologic culture of tissue of the osseous bulla and determination of antimicrobial susceptibility are recommended. Administration of cefazolin alone may not be efficacious for antimicrobial prophylaxis.     

Pyrenophora japonica occurring on barley in South Africa

A Pyrenophora teleomorph was recently found on barley stubble in South Africa. Single-ascospore isolates, mated on water agar containing sterile pieces of barley stubble, produced the teleomorph within 12 months of incubation at 15°C in the dark. Based on morphological characteristics and symptom expression on barley plants, the teleomorph is ascribed to P. japonica (anamorph Drechslera tuberosa ). This is the first report describing the occurrence of the teleomorph of D. tuberosa in South Africa.     

Antimicrobial Activity of Culture Filtrate of Bacillus amyloliquefaciens RC-2 Isolated from Mulberry Leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.