Bio-Sulfur Pre-Treatment Suppresses Anthracnose on Cucumber Leaves Inoculated with Colletotrichum orbiculare

Bio-sulfur can be produced in the process of desulfurization from a landfill and collected by some microorganism such as Thiobacillus sp. as a sulfur element. In order to investigate practical use of bio-sulfur as an agent for controlling plant disease, in vitro antifungal activity of bio-sulfur was tested against Colletotrichum orbiculare known to cause cucumber anthracnose. Efficacy of bio-sulfur for suppressing anthracnose disease was also evaluated in vivo using cucumber leaves. Mycelial growth of C. orbiculare on medium containing bio-sulfur was inhibited. Disease severity of cucumber leaves pre-treated with bio-sulfur was significantly decreased compared to that of untreated ones. To illustrate how bio-sulfur could suppress anthracnose disease, structures of cucumber leaves infected with C. orbiculare were observed under a fluorescent microscope and a scanning electron microscope (SEM). Cucumber leaves pre-treated with bio-sulfur showed a low rate of appressorium formation whereas untreated ones showed abundant appressoria. Shrunk fungal hyphae were mostly observed on bio-sulfur-pretreated leaves by SEM. Similar results were observed on leaves pre-treated with a commercial fungicide Benomyl®. These results suggest that inhibition of appressorium formation of C. orbiculare by bio-sulfur may contribute to its suppression of cucumber anthracnose.

Ultrastructures of Colletotrichum orbiculare in the Leaves of Cucumber Plants Expressing Induced Systemic Resistance Mediated by Glomus intraradices BEG110

The colonization of an arbuscular mycorrhizal fungus Glomus intraradices BEG110 in the soil caused a decrease in disease severity in cucumber plants after fungal inoculation with Colletotrichum orbiculare. In order to illustrate the resistance mechanism mediated by G. intraradices BEG110, infection patterns caused by C. orbiculare in the leaves of cucumber plants and the host cellular responses were characterized. These properties were characterized using transmission electron microscopy on the leaves of cucumber plants grown in soil colonized with G. intraradices BEG110. In the untreated plants, inter- and intra-cellular fungal hyphae were observed throughout the leaf tissues during both the biotrophic and necrotrophic phases of infection. The cytoplasm of fungal hyphae appeared intact during the biotrophic phase, suggesting no defense response against the fungus. However, several typical resistance responses were observed in the plants when treated with G. intraradices BEG110 including the formation of sheaths around the intracellular hyphae or a thickening of host cell walls. These observations suggest that the resistance mediated by G. intraradices BEG110 most often occurs in the symplast of the host cells rather than in the apoplast. In addition, this resistance is similar to those mediated by biotic inducers such as plant growth promoting rhizobacteria.

Resistance Induction and Enhanced Tuber Production by Pre-inoculation with Bacterial Strains in Potato Plants against Phytophthora infestans

Efficacy of resistance induction by the bacterial isolates Pseudomonas putida (TRL2-3), Micrococcus luteus (TRK2-2) and Flexibacteraceae bacterium (MRL412), which were isolated from the rhizosphere of plants growing in Jeju Mountain, were tested in a greenhouse. The disease severity caused by Phytophthora infestans was effectively reduced in the potato plants pre-inoculated with bacterial isolates compared with those of the untreated control plants growing in a greenhouse. In order to estimate the level of protection by the bacterial isolates, Mancozeb WP (Diesen M(R), Kyong nong) and DL-3-amino butyric acid (BABA) were pre-treated, whereas Dimethomorph WP (Forum(R), Kyong nong) and phosphonic acid (H3PO3) were post-treated the challenge inoculation with the pathogen. Disease severities of chemical pre-treated as well as post-treated plants were reduced compare to those of the untreated. The disease reduction in the plants pre-treated with Mancozeb WP was the highest, whereas that of post-treated with Dimethomorph WP was the lowest. The yields of plants pre-inoculated with three bacterial isolates were greatly increased than those of control plants. These results suggest that biological control by bacterial isolates might be an alternative strategy against late blight disease in potato plants growing in greenhouse.

Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 x 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 x 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.

Isolation of Rhizobacteria in Jeju Island Showing Anti-Fungal Effect against Fungal Plant Pathogens

To select active bacterial strains to control plant diseases, 57 bacterial strains were isolated from the rhizosphere of the plants growing in various areas such as coast, middle and top of Halla Mountain in Jeju Island. Anti-fungal effect of isolated bactrial strains was tested in vitro by incubating in potato dextrose agar with isolates of four fungal plant pathogens Rhizoctonia solani, Fusarium oxysporum, Colletotrichum gloeosporioides and C. orbiculare, respectively. Thirty-four bacterial strains inhibited the hyphal growth of the plant pathogens, from which 17 strains inhibited one of the tested fungi, 10 strains two fungi, six strains three and a strain TRL2-3 inhibited all of the tested fungi. Some bacterial strains could inhibit weakly the hyphal growth of the plant pathogens, whereas some did very strongly with apparent inhibition zone between the plant pathogens and bacterial strains indicating the unfavorable condition for hyphal growth. Although there was no apparent inhibition zone, some bacterial strains showed a strong suppression of hyphal growth of plant pathogens. Especially, the inhibition by TRL2-3 was remarkably strong in all cases of the tested plant pathogens in this study that could be a possible candidate for biological control of various plant diseases.

Selection and Efficacy of Soil Bacteria Inducing Systemic Resistance Against Colletotrichum orbiculare on Cucumber

Soil bacteria were screened for the ability to control cucumber anthracnose caused by Colletotrichum orbiculare through induced systemic resistance (ISR). Sixty-four bacterial strains having in vitro antifungal activity were used for selecting ISR-inducing strains in cucumber. Cucumber seeds (cv. Baeknokdadagi) were sown in potting mixtures incorporated with the soil bacteria, at a rate of ca. 10(8) cells per gram of the mixture. Two week-old plants were then transplanted into the steam-sterilized soil. Three leaf-stage plants were inoculated with a conidial suspension (5x10(5) conidia/ml) of C. orbiculare. Diseased leaf area (%) and number of lesions per cm2 leaf were evaluated on third leaves of the plants, 5~6 days after inoculation. Among 64 strains tested, nine strains, GC-B19, GC-B35, GK-B18, MM-B22, PK-B14, RC-B41, RC-B64, RC-B65, and RC-B77 significantly (P = 0.05) reduced anthracnose disease compared to the untreated control. In contrast, some bacterial strains promoted susceptibility of cucumber to the disease. From the repeated experiments using the nine bacterial strains, GC-B19, MM-B22, PK-B14, and RC-B65 significantly (P = 0.05) reduced both diseased leaf area (%) and number of lesions per cm2 leaf in at lease one experiment. These strains with control efficacy of 37~80% were determined to be effective ISR-inducing strains.

Different Mechanisms of Induced Systemic Resistance and Systemic Acquired Resistance Against Colletotrichum orbiculare on the Leaves of Cucumber Plants

Defense mechanisms against anthracnose disease caused by Colletotrichum orbiculare on the leaf surface of cucumber plants after pre-treatment with plant growth promoting rhizobacteria(PGPR), amino salicylic acid(ASA) or C. orbiculare were compared using a fluorescence microscope. Induced systemic resistance was mediated by the pre-inoculation in the root system with PGPR strain Bacillus amylolquefaciens EXTN-1 that showed direct antifungal activity to C. gloeosporioides and C. orbiculare. Also, systemic acquired resistance was triggered by the pre-treatments on the bottom leaves with amino salicylic acid or conidial suspension of C. orbiculare. The protection values on the leaves expressing SAR were higher compared to those expressing ISR. After pre-inoculation with PGPR strains no change of the plants was found in phenotype, while necrosis or hypersensitive reaction(HR) was observed on the leaves of plants pre-treated with ASA or the pathogen. After challenge inoculation, inhibition of fungal growth was observed on the leaves expressing both ISR and SAR. HR was frequently observed at the penetration sites of both resistance-expressing leaves. Appressorium formation was dramatically reduced on the leaves of plants pre-treated with ASA, whereas EXTN-1 did not suppress the appressorium formation. ASA also more strongly inhibited the conidial germination than EXTN-1. Conversely, EXTN-1 significantly increased the frequency of callose formation at the penetration sites, but ASA did not. The defense mechanisms induced by C. orbiculare were similar to those by ASA. Based on these results it is suggested that resistance mechanisms on the leaf surface was different between on the cucumber leaves expressing ISR and SAR, resulting in the different protection values.