Occurrence and Detection of Carbendazim Resistance in Botryosphaeria dothidea from Apple Orchards in China.

Botryosphaeria dothidea causes white rot, which is among the most devastating diseases affecting apple crops globally. In this study, we assessed B. dothidea resistance to carbendazim by collecting samples from warts on the infected branches of apple trees or from fruits exhibiting evidence of white rot. All samples were collected from different orchards in nine provinces of China in 2018 and 2019. In total, 440 B. dothidea isolates were evaluated, of which 19 isolates from three provinces were found to exhibit carbendazim resistance. We additionally explored the fitness and resistance stability of these isolates, revealing that they were no less fit than carbendazim-sensitive isolates in terms of pathogenicity, sporulation, and mycelial growth and that the observed carbendazim resistance was stable. Sequencing of the ß-tubulin gene in carbendazim-resistant isolates showed the presence of a substitution at codon 198 (GAG to GCG) that results in an alanine substitution in place of glutamic acid (E198A) in all 19 resistant isolates. A loop-mediated isothermal amplification (LAMP) method was then developed to rapidly and specifically identify this E198A mutation. This LAMP method offers value as a tool for rapidly detecting carbendazim-resistant isolates bearing this E198A mutation and can thus be used for the widespread monitoring of apple crops to detect and control the development of such resistance.

Identification and Detection of Botryosphaeria dothidea from Kiwifruit (Actinidia chinensis) in China.

Kiwifruit is very popular among consumers due to its high nutritional value. The increasing expansion in kiwifruit cultivation has led to the spread of rot diseases. To identify the pathogens causing kiwifruit ripe rots in China, 24 isolates were isolated from the diseased fruit and wart in trees. Botryosphaeria dothidea was recognized as the pathogen causing kiwifruit ripe rot and wart in the tree through internal transcribed spacer (ITS) sequencing, pathogenicity testing, morphological and microscopic characteristics. The rapid and accurate detection of this pathogen will lead to better disease monitoring and control efforts. A loop-mediated isothermal amplification (LAMP) method was then developed to rapidly and specifically identify B. dothidea. These results offer value to further research into kiwifruit ripe rot, such as disease prediction, pathogen rapid detection, and effective disease control.

Agrobacterium tumefaciens-mediated transformation of Coniella granati.

Pomegranate fruit rot caused by Coniella granati is among the most devastating diseases threatening pomegranate production. The pathogenic mechanism of this pathogen remains largely unknown due to lack of genetic transformation method. Herein, we developed an approach to the Agrobacterium tumefaciens-mediated transformation (ATMT) of C. granati using a plasmid vector encoding the green fluorescent protein (GFP) and hygromycin resistance (Hyg) genes. This approach yielded C. granati transformants that exhibited uniform, stable green fluorescence. We further optimized this ATMT protocol, enabling us to achieve a transformation efficiency of up to 300 transformants per 0.5 cm2 mycelial plug. Together, we thus provide the first report of the stable transformation of C. granati, laying a foundation for future functional studies characterizing this economically important fungal pathogen.

Isolation and Characterization of Bacillus velezensis Strain P2-1 for Biocontrol of Apple Postharvest Decay Caused by Botryosphaeria dothidea.

Botryosphaeria dothidea causes apple ring rot, which is among the most prevalent postharvest diseases of apples and causes significant economic loss during storage. In this study, we investigated the biocontrol activity and possible mechanism of Bacillus velezensis strain P2-1 isolated from apple branches against B. dothidea in postharvest apple fruit. The results showed strain P2-1, one of the 80 different endophytic bacterial strains from apple branches, exhibited strong inhibitory effects against B. dothidea growth and resulted in hyphal deformity. B. velezensis P2-1 treatment significantly reduced the ring rot caused by B. dothidea. Additionally, the supernatant of strain P2-1 exhibited antifungal activity against B. dothidea. Re-isolation assay indicated the capability of strain P2-1 to colonize and survive in apple fruit. PCR and qRT-PCR assays revealed that strain P2-1 harbored the gene clusters required for biosynthesis of antifungal lipopeptides and polyketides. Strain P2-1 treatment significantly enhanced the expression levels of pathogenesis-related genes (MdPR1 and MdPR5) but did not significantly affect apple fruit qualities (measured in fruit firmness, titratable acid, ascorbic acid, and soluble sugar). Thus, our results suggest that B. velezensis strain P2-1 is a biocontrol agent against B. dothidea-induced apple postharvest decay. It acts partially by inhibiting mycelial growth of B. dothidea, secreting antifungal substances, and inducing apple defense responses.