Barcode-like heteroduplex DNA pattern as an aid for rapid identification of anthracnose fungi

We have shown the usefulness of the heteroduplex mobility assay (HMA) for phylogenetic analysis and for the discrimination of closely related Colletotrichum species. Because the heteroduplex mobility of a tested strain shows a unique banding pattern that is the function of the sequence of the referred strain, we further explored the potential use of heteroduplex DNA patterns (HPs) as DNA fingerprints for the identification of these fungi. The 29 Colletotrichum strains previously identified by HMA to be taxonomic members of CG, CA, CM, CC and CL species groups were re-examined with an emphasis on their unique heteroduplex banding patterns. The species attributes of these tested strains were characterized by HMA using ITS fragments amplified from six representative Colletotrichum strains as pairwise compared references. By comparing the unique homoduplex and heteroduplex banding patterns of each tested strain on a polyacrylamide gel with those of the respective reference strain, the species identity of tested strains was determined. The obtained barcode-like HPs classified these 29 Colletotrichum strains into 6 distinctive groups: CG1, CG2, CA, CM, CC and CL. Notably, the HPs differentiated strains CG1 and CG2, which differed in their ITS sequences by only six bases. The presented results revealed that the species-characteristic barcode-like HP classification of ITS regions is a relatively rapid and valuable system for species identification of Colletotrichum species. The potential use of the established barcode-like system for the identification of anthracnose fungi and other fungal pathogens is discussed

DNA Polymorphisms and Biocontrol of Bacillus Antagonistic to Citrus Bacterial Canker with Indication of the Interference of Phyllosphere Biofilms

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S–23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease

Bacterial strains and plasmids used in this study.

a<p>Species attributes of native <i>Bacillus</i> strains were identified based on Biolog analysis except for strain OF3-16, which was identified based on physiological and biochemical characteristics described in the Bergey's Manual of Systematic Bacteriology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042124#pone.0042124-Sneath1" target="_blank">[39]</a>.</p>b<p>ATCC, American Type Culture Collection Center; BCRC, Bioresource Collection and Research Center, Taiwan; DSM, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; NCBI, National Center for Biotechnology Information.</p

Antagonistic activity of <i>Bacillus</i> species against <i>X. axonopodis</i> pv. <i>citri</i> XW19.

<p>A 20 µl aliquot of <i>X. axonopodis</i> pv. <i>citri</i> XW19 suspension (OD₆₂₀ = 0.3) was spread on an SYB agar plate. Following overnight incubation at 30°C, 20 µl of <i>Bacillus</i> suspension (OD₆₂₀ = 0.3) was spotted (A) inside the stainless steel ring or (B) on a paper disc. The plates were incubated at 30°C, for 5 days. CK, 20 µl of sterile Milli-Q water was used as a control. The results represent the means and standard deviations (error bars) of a representative experiment. Different lowercase letters indicate significant differences (p<0.05) according to Tukey's HSD test.</p

Phylogenetic tree of <i>Bacillus</i> species based on 16S rRNA gene sequences.

<p>The tree was constructed using the neighbor-joining method and genetic distances were generated using the Kimura 2-parameter method. The numbers at the branches are bootstrap confidence percentages from 1000 bootstrapped trees. <i>Alicyclobacillus acidocaldarius</i> (GenBank accession no. AB089859) was used as the outgroup. The numbers in parentheses indicate the GenBank accession numbers.</p

tDNA-PCR fingerprint and UPGMA cluster analysis of <i>Bacillus</i> species.

<p>(A) tDNA-PCR fingerprint and (B) UPGMA cluster analysis. The UPGMA cluster analysis was based on tDNA-PCR. M, GeneRuler™ 100 bp plus DNA ladder (Fermentas, Taipei, Taiwan).</p

The effect of application time and frequency of <i>B. subtilis</i> strain TKS1-1 on symptom development and disease incidence of citrus bacterial canker on navel orange grown in the greenhouse.

<p><i>B. subtilis</i> strain TKS1-1 endospore formulation (T, 10⁹ CFU/ml) and <i>X. axonopodis</i> pv. <i>citri</i> XW19 (B, 10⁸ CFU/ml) were used. Treatment T-B-T/1WK, strain TKS1-1 endospores were applied 24 h prior to inoculation with <i>X. axonopodis</i> pv. <i>citri</i> XW19, and then weekly post-pathogen inoculation for 4 weeks; treatment T-B-D/1WK, strain TKS1-1 endospores were applied 24 h prior to <i>X. axonopodis</i> pv. <i>citri</i> XW19 inoculation; treatment D-B-T/1WK, strain XW19 was inoculated, then strain TKS1-1 endospores were sprayed every week post-pathogen inoculation for 4 weeks; treatment D-B-D/1WK, only strain XW19 was inoculated. Without <i>Bacillus</i> treatment, Milli-Q water (D) was sprayed on the leaf surface. (A) Symptoms on upper (left) and lower (right) leaf surfaces after different treatments. Scale bars, 1 cm. (B) Disease incidence was rated 4 weeks post-inoculation. Bars indicate standard deviations. Columns that are top-labeled with different letters are significantly different (p<0.05) according to one-way ANOVA and Tukey's HSD test.</p

ITS-PCR fingerprint and UPGMA cluster analysis of <i>Bacillus</i> species.

<p>(A) ITS-PCR fingerprint and (B) UPGMA cluster analysis. The UPGMA cluster analysis was based on ITS-PCR. M, GeneRuler™ 100 bp plus DNA ladder (Fermentas, Taipei, Taiwan).</p