Blackleg is a worldwide disease of canola (Brassica napus), caused by a complex of fungal species in the genus Leptosphaeria, that impacts canola production and seed quality. Demethylation inhibitor (DMI) fungicides that target sterol 14α-demethylase are an integral part of disease control. Here, we report six DMI-resistant isolates of Leptosphaeria maculans and two different types of genetic modification related to the resistance. Analysis of the regulatory region of the DMI target gene ERG11 (also known as CYP51) revealed a 275-bp insertion in two of the isolates and three long terminal repeat retrotransposons (5,263, 5,267, and 5,248 bp) inserted in the promoter region of three resistant isolates. Genetic approaches confirmed that these elements are responsible for DMI resistance in L. maculans and crosses show segregation consistent with a single locus. Reverse-transcription quantitative PCR assays demonstrated that the 275-bp insertion increases ERG11 gene expression, conferring DMI fungicide resistance both in vitro and in planta. Moreover, transformation of a susceptible isolate of L. maculans with ERG11 driven by a promoter containing the 275-bp insertion increased resistance to tebuconazole. A minimal shift of the values of concentration whereby 50% of the mycelial growth is inhibited in vitro was observed in resistant isolates containing long terminal repeat retrotransposons; nevertheless, these isolates were able to develop significant lesions on cotyledons from fungicide-treated seedlings. This is the first report of genetic modifications in L. maculans relating to DMI fungicide resistance.

Leptosphaerulina leaf spot, caused by Leptosphaerulina trifolii, is a major disease of alfalfa (Medicago sativa), leading to noticeable losses. From 2022 to 2023, we collected samples of alfalfa with symptoms of the disease from different locations in Inner Mongolia, China. Nine fungal isolates recovered from these samples were identified through morphological traits and a maximum likelihood phylogeny based on concatenated partial sequences of ITS, 28S, and rpb2. A pathogenicity test on alfalfa confirmed the pathogenicity of the isolates on alfalfa. Analysis of physiological traits of L. trifolii revealed optimal mycelium growth at 20 °C and a pH range of 5 to 7, with soluble starch as the preferred carbon source and yeast extract as the optimal nitrogen source. The pathogen thrived in V8-juice agar and oat agar media. This study confirms L. trifolii as the causative agent of Leptosphaerulina leaf spot of alfalfa in Inner Mongolia and provides valuable insights into its optimal growth conditions. These findings enhance the understanding and management of this disease in alfalfa fields.

Continuous cropping of Brassica napus impairs sustainable production via soil nutrient imbalance and microecological degradation. We evaluated rhizosphere soil properties and microbial communities under rotations crops (Triticum aestivum [TaBn], Beta vulgaris [BvBn], Glycine max [GmBn], Sorghum bicolor [SbBn], Hordeum vulgare [HvBn], and Brassica napus [BnBn]). BvBn had the highest total nitrogen, total potassium, available potassium, and organic matter contents. TaBn exhibited the highest soil enzyme activities, and its bacterial/fungal Chao1/Simpson indices and unique operational taxonomic units (OTUs; bacteria: 333, fungi: 37) exceeded other patterns. Principal coordinate analysis showed distinct microbial community separation in BvBn/TaBn versus BnBn. TaBn enriched dominant bacterial phyla Pseudomonadota and Actinomycetota; all preceding crops increased fungal phylum Ascomycota while reducing Mucoromycota. Comprehensive assessment confirmed all preceding crops, except oilseed rape altered rhizosphere microbial structure, with TaBn as the optimal preceding crops.

Fruit rot is one of the major diseases impacting the production and quality of zucchini (Cucurbita pepo). In August 2021, fruit rot symptoms were observed on the zucchini fruit ‘Jindi 1’ in the Wuyuan region in Inner Mongolia, China with an incidence ranging from 10% to 30%. Where the pepo was in contact with the soil, dark grey and spongy corky lesions 4–5 cm in diameter with a light brown halo were observed. The internal necrosis of the fruit rind was also recorded. From the affected fruits, fungal colonies belonging to the Fusarium species were exclusively isolated. Molecular analysis of the ITS, TEF-1α, and RPB2 sequences identified the isolates as Fusarium solani f. sp. cucurbitae. Inoculated on ‘Jindi 1’, the strain Fx-1a induced typical fruit rot on the pepo and wilting on seedlings, while negative-controls remained asymptomatic. The impact of this disease on seed quality and yield in zucchini seed production needs to be further studied.

Alternative splicing (AS) is a prevalent post-transcriptional regulatory mechanism in eukaryotes and plays a crucial role in plant disease resistance. Here, we used the Illumina Novaseq sequencing platform to conduct transcriptome sequencing on canola (Brassica napus) leaves infected with the blackleg pathogen (Leptosphaeria biglobosa strain nm⁻¹) at 0 h, 72 h, 120 h, and 168 h post-inoculation to investigate the mechanism of AS coordination with transcriptional regulation in canola’s response to blackleg disease. The rMATS software (4.1.0) was employed to analyze different AS events in samples taken at 72 h, 120 h, and 168 h. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to elucidate the biological functions of differentially spliced genes at various time points, while Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify key modules and hub genes. As a result, our analysis reveals 16908 AS events across three time points, with 221 being differently spliced. Intron retention (RI) was the most common AS event, accounting for approximately 55% of all events, while alternative 5′ splice site events were least common, comprising only 2%. Furthermore, a total of 213 significantly differentially spliced genes were identified, which were enriched in functions related to protein kinase activity, transferase activity, and pathways such as MAPK signaling pathway—plant and plant hormone signal transduction. WGCNA identified three key modules and ten hub genes, including calcium-binding transcription activator 1, LRR class receptor serine/threonine protein kinase FEI 2, PLATZ transcription factor family proteins, serine/threonine protein kinase PRP4, and E3 ubiquitin ligase SUD1, all of which are associated with canola resistance to L. biglobosa. Thus, this study provides a theoretical basis for identifying disease-resistance genes involved in AS and for exploring the functions of AS gene isoforms in canola.

Zucchini (Cucurbita pepo L.) is an important crash crop for producing seeds in Inner Mongolia, China. In August 2021, the harvest season of seed-producing zucchini, fruit rot symptom (cv. Jindi No.1) was found in the Wuyuan region. The incidence of infected fruits of zucchini ranged from 10% to 30% and symptom consisted in 4 to 5 cm diameter lesions where the fruit was in contact with the soil. Ten isolations from six symptomatic fruits were preliminary classified as Fusarium species based on its morphological characters in potato dextrose agar. Phylogenetic tree was constructed by concatenating TEF-1α and RPB2 gene and exhibited the isolates clustered together with the Fusarium solani f. sp. cucurbitae sequences. Pathogenicity tests revealed that two isolates caused over 3 cm diameter lesions on the fruits while negative-controls remained asymptomatic. The colonies reisolated from the leasions were morphologically identical to the original isolates, thus fulfilling Koch’s postulates. Results demonstrated that F. solani f. sp. cucurbitae was the causal agent of fruit rot observed in the field. To our knowledge, this is the first report of fruit rot of seed-producing zucchini caused by F. solani f. sp. cucurbitae in Inner Mongolia, China.