Biocontrol Ability and Mechanism of a Broad-Spectrum Antifungal Strain Bacillus safensis sp. QN1NO-4 Against Strawberry Anthracnose Caused by Colletotrichum fragariae

Strawberry is a very popular fruit with a special taste, color, and nutritional value. Anthracnose caused by Colletotrichum fragariae severely limits fruit shelf life during post-harvest storage. Use of traditional chemical fungicides leads to serious environment pollution and threatens food safety. Biocontrol is considered as a promising strategy to manage the post-harvest fruit diseases. Here, strain QN1NO-4 isolated from noni (Morinda citrifolia L.) fruit exhibited a high antifungal activity against C. fragariae. Based on its physicochemical profiles and phylogenetic tree of the 16S rRNA sequence, strain QN1NO-4 belonged to the genus Bacillus. The average nucleotide identity (ANI) calculated by comparing two standard strain genomes was below 95–96%, suggesting that the strain might be a novel species of the genus Bacillus and named as Bacillus safensis sp. QN1NO-4. Its extract effectively reduced the incidence of strawberry anthracnose of harvested fruit. Fruit weight and TSS contents were also maintained significantly. The antifungal mechanism assays indicated that the extract of the test antagonist inhibited mycelial growth and spore germination of C. fragariae in vitro. Cells of strain QN1NO-4 demonstrated the cytoplasmic heterogeneity, disappeared organelles, and ruptured ultrastructure. Notably, the strain extract also had a broad-spectrum antifungal activity. Compared with the whole genome of strain QN1NO-4, several functional gene clusters involved in the biosynthesis of active secondary metabolites were observed. Fifteen compounds were identified by gas chromatography–mass spectrometry (GC-MS). Hence, the fruit endophyte B. safensis sp. QN1NO-4 is a potential bio-agent identified for the management of post-harvest disease of strawberry fruit.

Design, Synthesis and Biological Evaluation of N-((2-phenyloxazol-4-yl)methyl) Pyrimidine Carboxamide Derivatives as Potential Fungicidal Agents

Twelve N-((2-phenyloxazol-4-yl)methyl) pyrimidine carboxamide derivatives were designed, synthesized, and characterized by 1H NMR, 13C NMR, and HRMS. The fungicidal activities of these new compounds against Sclerotinia sclerotiorum, Botrytis cinereal, and Colletotrichum fragariae were evaluated. The results indicated that compounds 5b, 5f, and 5g displayed potential fungicidal activities against tested fungi, especially 5f exhibited IC50 value of 28.9 mg/L against S. sclerotiorum. Moreover, the compounds 5f and 5g showed IC50 values of 54.8 mg/L and 62.2 mg/L against C. fragariae respectively, which shows that they were more active than the commercial fungicide hymexazol. The superficial structure-activity relationships were discussed, which may be of benefit for the development of fungicides and discovery of novel fungicides.

Phytotoxic Lignans from Artemisia arborescens

A systematic bioassay-guided fractionation of methylene chloride extracts of the aerial part of Artemisia arborescens was performed in order to identify its phytotoxic compounds Two lignans were isolated, sesamin and ashantin, that inhibited growth of Agrostis stolonifera (bentgrass), a monocot, and Lactuca sativa (lettuce), a dicot, at 1 mg mL–1. In a dose-response screening of these lignans for growth inhibition against Lemna paucicostata (duckweed), ashantin was the most active with an IC50 of ca. 224 μM. The mode of action of these compounds is still unknown. In mosquito larvicidal bioassays the pure compounds sesamin and ashantin did not cause mortality at the highest dose of 125 mg/L against 1-d-old Aedes aegypti larvae. In bioautography bioassays for antifungal activity using Botrytis cinerea, Fusarium oxysporum, Colletotrichum fragariae, Colletotrichum acutatum, and Colletotrichum gloeosporioides, ashantin and sesamin were inactive at 5 μg and were therefore not subjected to additional screening in secondary antifungal assays.

First Report of Anthracnose Caused by Colletotrichum fragariae on Cyclamen in North Carolina

In November 2009, cyclamen (Cyclamen persicum) plants with disease symptoms from a commercial greenhouse operation in the western part of North Carolina were sent to the Plant Diseases and Insect Clinic at North Carolina State University. Symptoms consisted of coalescing reddish and tan necrotic leaf spots with concentric circles. Other symptoms included darkened vascular tissue and decay of the corm, large roots, and petioles. Diseased leaves and stems were surface sterilized in 0.5% sodium hypochlorite for 3 min, air dried, and placed in petri dishes containing alkaline water agar. After 3 days of incubation at room temperature, fungal colonies were transferred to acidified potato dextrose agar. Isolation frequency after 5 days was 33% (three of nine pieces) and 16% (one of six pieces) from small leaf spots and petioles, respectively. Pure cultures of isolates were gray and black with abundant, aerial, gray whitish mycelia. Diseased plants were also incubated in a moist chamber at room temperature and sporulation was observed within 7 days. Conidia were tapered with rounded ends and produced in the acervulus and on the tips of setae, which is consistent with the morphology of described isolates of Colletotrichum fragariae. Similar setae were also observed directly on the fine roots of the original sample. The pathogenicity of single-spore cultures was tested by spraying four 2-month-old cyclamen plants with a conidial suspension (106 conidia/ml) and the plants were kept in a humid chamber for 24 h. Noninoculated controls (four plants) were sprayed with distilled water and subjected to the same conditions. The pathogenicity test was also repeated. Inoculated plants and controls were placed in a greenhouse with a temperature range from 22 to 25°C. After 7 to 10 days, symptomatic leaves and stems were observed on all the inoculated plants but not on the control plants. Fungi reisolated from 10 symptomatic leaf tissues had identical morphological features as the original isolates. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits following the manufacturer's protocol (Qiagen Inc., Valencia, CA). Sequence analysis of the rRNA internal transcribed spacer (ITS) region of the cyclamen isolate (GenBank Accession No. HQ188923), based on the fragment amplified with ITS1 and ITS4 primers, showed 100% similarity to isolates of C. fragariae deposited in GenBank (Accession Nos. FJ172290 [ATCC MYA-4443 from cyclamen] and FJ810510 [ATCC MYA-4442 from silver date palm]) and Florida isolate C16 isolated from strawberry (1). In addition, the morphology and ITS sequences of the cyclamen isolate were identical to those of the C. fragariae voucher isolate from strawberry (GU174546). Results from disease symptoms, colony and spore morphology, pathogenicity tests, and ITS sequence analysis suggest that C. fragariae was the pathogen responsible for the disease symptoms on cyclamens. To our knowledge, this is the first report of a disease caused by C. fragariae on cyclamen in North Carolina and complements an earlier report from Florida (1). Reference: (1) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008.