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.

Biological characteristics of the strawberry anthracnose pathogen Colletotrichum nymphaeae and methods of its diagnostics

This work presents the research results of artificial infection of garden strawberry plants of the Elsanta variety with the phytopathogenic fungus Colletotrichum nymphaeae from the Colletotrichum acutatum complex. Three different ways of infecting strawberry plants were analysed: by injecting the plant rosette, spraying with a spore suspension, and infecting the soil. The study showed that the plants of this variety are susceptible to the C. nymphaeae species. The most susceptible parts of plants were identified: leaf blades, petioles and strawberry rosettes. The most obvious symptoms of infection, caused by C. nymphaeae, were found in case of injecting into the rosette. In this work, the most sensitive and valid laboratory methods for isolating and identifying the causative agent of strawberries anthracnose on the planting material were determined: isolation of the pathogen using the deposit method on the 2 % PGA nutrient medium with the addition of 4 % citric acid and identification by the real-time PCR method using the “Fitoscreen set. C. acutatum complex-PB” (CJSC Syntol, Moscow). The C. nymphaeae species from the C. acutatum complex is a pathogen harmful to strawberry plants, causing rapid damage to leaf blades, petioles and rosettes. In the conducted experiment, the root system of strawberry plants was not affected, and there was no infection in the soil.

Identification of Colletotrichum species associated with anthracnose disease of strawberry in Sichuan Province, China

Strawberry anthracnose, caused by Colletotrichum species, is a major fungal disease threatening strawberry industry in Sichuan Province of southwestern China. However, research on identification of Colletotrichum species associated with strawberry anthracnose in Sichuan remains uncovered. In this study, 73 representative Colletotrichum strains were isolated from diseased leaves, stolons, petioles and crowns of 11 major strawberry planting localities in Sichuan Province. Based on morphological characteristics and multi-loci phylogenetic analysis, the Colletotrichum strains were identified as three distinct species: Colletotrichum fructicola (53 strains, 72.60%), C. siamense (17 strains, 23.29%) and C. gloeosporioides sensu stricto (3 stains, 4.11%). Among them, C. fructicola was the most ubiquitous and dominant species, whereas, C. gloeosporioides sensu stricto only restricted in Chongzhou. Importantly, our pathogenicity tests showed that C. fructicola and C. siamense can infect both leaves and stolons, while C. gloeosporioides sensu stricto was only pathogenic to leaves. Interestingly, although the sexual stage of C. siamense was not observed in this study, it still exhibited the strongest virulence to strawberry as compared to C. gloeosporioides sensu stricto and C. fructicola. Overall, this is the first study to characterize Colletotrichum species causing strawberry anthracnose and evaluate their pathogenicity in Sichuan Province of southwestern China, which will provide a better strategy on accurate diagnosis and management on anthracnose disease on strawberry.

Characterization of Colletotrichum spp. Sensitivity to Carbendazim for Isolates Causing Strawberry Anthracnose in China

Strawberry anthracnose caused by Colletotrichum species is an important disease that may cause significant economic losses. Based on multilocus sequence analyses and morphological characteristics, 64 isolates from strawberry anthracnose samples collected from nine Chinese provinces and municipalities were identified as three species: Colletotrichum fructicola (29 isolates), Colletotrichum siamense (23 isolates), and Colletotrichum nymphaeae (12 isolates). Isolates of C. siamense showed strong aggressiveness to fruit and leaves. Isolates of C. fructicola showed strong aggressiveness to crowns. Isolates of C. nymphaeae were weakly or not pathogenic to fruit, leaves, or crowns. Sensitivity to carbendazim was determined for a total of 75 isolates, including 11 previously preserved. Two isolates of C. siamense were highly resistant (HR) and 21 were moderately resistant (MR). Nine isolates of C. fructicola were sensitive (S), 24 were HR, and four were MR. All 15 isolates of C. nymphaeae were insensitive, and their mycelial growth was not completely inhibited on potato dextrose agar amended with 500 μg/ml carbendazim. Beta-tubulin (TUB2) of representative isolates was amplified and sequenced, revealing a glutamic acid substituted by alanine at codon 198 in HR isolates of C. siamense and C. fructicola. MR isolates of C. siamense and C. fructicola had a point mutation at codon 200, causing a replacement of phenylalanine acid by tyrosine. No point mutation was detected at codons 50, 167, 198, 200, or 240 in TUB2 of C. nymphaeae insensitive isolates. Overall, this study revealed that C. fructicola was the dominant species causing anthracnose on strawberry and could improve the understanding of the management of fungicide resistance in Colletotrichum species on strawberry in China.

Resistance to azoxystrobin and thiophanate-methyl is widespread in Colletotrichum spp. isolates from the Mid-Atlantic Strawberry Fields

Multiple Colletotrichum species have been found to be responsible for strawberry anthracnose, and prevalence of each species seems to vary by regions and/or host tissues. In this study, a total of 200 Colletotrichum isolates were obtained from different strawberry cultivars displaying anthracnose symptoms in the Mid-Atlantic fields. Analysis of g3pdh, tub2, and/or ITS sequences revealed four Colletotrichum species, including C. nymphaeae, C. fioriniae, C. siamense, and C. lineola. C. nymphaeae was the predominant species, representing 90% of all isolates collected. This species was found from all strawberry organs/tissues examined, whereas C. siamense and C. fioriniae were limited to the crown and fruit, respectively. Further, all Colletotrichum isolates were screened for resistance to azoxystrobin in vitro, and all C. siamense isolates were additionally screened for resistance to thiophanate-methyl. The overall frequency of resistance to azoxystrobin and thiophanate-methyl was 48.0 % and 67.0 %, respectively. G143A in the cytochrome b gene (cyt b) was found in all C. nymphaeae and C. siamense isolates with high level of resistance, with EC50 > 100 µg/ml, while F129L was found in two of the five C. nymphaeae isolates with moderate resistance, with EC50 values ranging from 2.6 to 7.8 µg/ml. All C. fioriniae isolates tested were found to be less sensitive to azoxystrobin, with EC50 values ranging from 9.7 to 14.4 µg/ml, despite no mutations detected in cyt b. Moreover, E198A in tub2 was linked with C. siamense isolates resistant to thiophanate-methyl (EC50 > 100 µg/ml). These results revealed that resistance in Colletotrichum spp. to primary fungicides was widespread in the Mid-Atlantic strawberry fields.