Coccinonectria pachysandricola, Causal Agent of a New Foliar Blight Disease of Sarcococca hookeriana.

Woody plants of the Buxaceae, including species of Buxus, Pachysandra, and Sarcococca, are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on S. hookeriana at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to P. terminalis exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as Coccinonectria pachysandricola (Nectriaceae, Hypocreales), causal agent of Volutella blight of Pachysandra species. Pathogenicity tests established that Co. pachysandricola isolated from both hosts caused disease symptoms on P. terminalis and S. hookeriana, but not on B. sempervirens. Artificial inoculations with Pseudonectria foliicola, causal agent of Volutella blight of B. sempervirens, did not result in disease on P. terminalis or S. hookeriana. Wounding enhanced infection by Co. pachysandricola and Ps. foliicola on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: Co. pachysandricola, Ps. buxi, and Ps. foliicola; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of S. hookeriana represents a new disease for this host and is capable of causing considerable damage to infected plants.

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer.

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.

Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae.

Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare. The 16S rRNA gene sequences of F. columnare (eight sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F. columnare and do not have significant intraspecies variability. The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR. The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F. columnare and there was no amplification in the closely related species. The specificity of the amplified product was confirmed by digesting with HhaI. The PCR primers did not produce a 675 bp product with F. columnare ATCC43622 strain. This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium johnsoniae. The American Type Culture Collection has confirmed these findings and made the change.

Inhibition of Germination, Outgrowth, and Vegetative Growth of Clostridium botulinum 67B by Spice Oils.

The oils of clove, thyme, black pepper, pimenta, origanum, garlic, onion, and cinnamon were tested for their activity on germination, outgrowth, and vegetative growth of Clostridium botulinum 67B in broth media. Garlic, onion, cinnamon, thyme, origanum, and black pepper oils at a concentration of 100 ppm prevented germination of C. botulinum 67B spores. Clove and pimenta oils at a concentration of 150 ppm prevented germination. The effect of spice oils on spore germination was reversible. The oils of black pepper and clove had a greater inhibitory effect on vegetative growth than the other oils. None of the oils had a significant effect on outgrowth.

Effect of Sodium Nitrite and Origanum Oil on Growth and Toxin Production of Clostridium botulinum in TYG Broth and Ground Pork.

Inhibition of Clostridium botulinum (strains A, B, and E) growth and toxin production by various concentrations of origanum oil and sodium nitrite was studied for both TYG broth and a model meat system. At concentrations of 100 and 200 ppm, origanum oil was effective in inhibiting C. botulinum growth in TYG broth. In addition, origanum oil acted synergistically with sodium nitrite in inhibiting C. botulinum growth in TYG broth. The inhibitory effect of origanum oil in the meat system was dramatically reduced and had a significant (p≤ 0.05) effect only when used at 400 ppm in combination with 50-100 ppm sodium nitrite.