Diversity of Lasiodiplodia Species Associated with Canker and Dieback in Fruit Trees in the Henan and Shandong Provinces of China.

Lasiodiplodia is a widely distributed genus that is associated with a variety of diseases in many plant species, especially fruit trees. In this study, a disease survey of fruit trees growing in 12 orchards located in the Henan and Shandong provinces of China was conducted between 2020 and 2022. The symptoms observed included stem canker, branch dieback, and gummosis. Phylogenetic analyses of internal transcribed spacer, tub2, tef1, and rpb2 sequence data combined with morphological characteristics revealed that the 19 isolates collected during the survey belonged to five documented Lasiodiplodia species, namely, Lasiodiplodia citricola, L. chiangraiensis, L. huangyanensis, L. pseudotheobromae, and L. theobromae, and two previously undescribed species, L. xinyangensis and L. ziziphi. In addition, the survey identified three novel host-pathogen interactions: L. chiangraiensis on loquat, L. citricola on apple, and L. huangyanensis on grapevine. Furthermore, the detailed phylogenic analysis indicated that four previously described Lasiodiplodia species were genetically very closely related that they would be better classified as synonyms rather than distinct species, so L. paraphysoides and L. nanpingensis should be considered synonyms of L. citricola, L. fujianensis should be a synonym of L. iraniensis, and L. henanica should be a synonym of L. huangyanensis. Pathogenicity tests confirmed that representative isolates of the two novel species and three new host-pathogen interactions identified in the current study were pathogenic to their original hosts, thereby fulfilling Koch's postulates. Similarly, all of the isolates were found to be pathogenic on four alternative hosts, although a high degree of variation in virulence was observed.

Molecular characterization of a novel ourmia­like virus from the phytopathogenic fungus Botryosphaeria dothidea.

Here, we describe a novel ourmia-like virus, Botryosphaeria dothidea ourmia-like virus 2 (BdOLV2), derived from the phytopathogenic fungus Botryosphaeria dothidea strain ZM180192-1 infecting maize in Henan province of China. The complete genome sequence of BdOLV2 consists of a positive-sense single-stranded RNA (+ ssRNA) segment with a length of 2,532 nucleotides (nt). The sequence contains a large open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) consisting of 605 amino acids (aa) with a molecular mass of 68.59 kDa. This RdRp protein contains eight typical conserved motifs associated with ourmia-like viruses. BLASTp analysis revealed that the RdRp protein of BdOLV2 had the highest similarity (62.10%, 58.15%, and 55.75% identity, respectively) to a virus previously identified as "Botourmiaviridae sp.", Macrophomina phaseolina ourmia-like virus 2, and Macrophomina phaseolina ourmia-like virus 2-A. Phylogenetic analysis based on the RdRp aa sequence indicated that BdOLV2 is a new member of the genus Magoulivirus in the family Botourmiaviridae.

Lasiodiplodia regiae sp. nov.: A New Species Causing Canker and Dieback of Fruit Trees in China.

Canker and dieback are serious fungal diseases of woody plants that can cause huge economic losses to orchards. The purpose of this study was to classify and assess the pathogenicity of fungal species associated with canker and dieback on fruit trees growing in Henan Province, China. In total, 150 isolates of Botryosphaeriaceae were obtained from six different fruit trees exhibiting typical symptoms of stem canker, branch dieback, and gummosis. Morphological examinations and phylogenetic analysis of ITS, tef1, tub2, and rpb2 revealed two Botryosphaeriaceae species, which are Botryosphaeria dothidea and a novel species, Lasiodiplodia regiae, respectively. Using Koch's postulates, we confirmed that the different isolates of L. regiae can cause disease in their original hosts. The pathogenicity tests showed that L. regiae can cause canker, dieback, and gummosis symptoms in four different hosts, indicating a relatively wider host range. Moreover, 10 L. regiae isolates exhibited similar symptoms but different levels of virulence on shoots of peach trees under field conditions. This study demonstrated that L. regiae was a new causal agent of canker and dieback of six fruit tree species, which could be a serious risk to the orchard industry in China. Furthermore, the findings provide a foundation for further epidemiological studies and the development of management strategies.

BsTup1 is required for growth, conidiogenesis, stress response and pathogenicity of Bipolaris sorokiniana.

Tup1, a conserved transcriptional repressor, plays a critical role in the growth and development of fungi. Here, we identified a BsTup1 gene from the plant pathogenic fungus Bipolaris sorokiniana. The expression of BsTup1 showed a more than three-fold increase during the conidial stage compared with mycelium stage. Deletion of BsTup1 led to decrease hyphal growth and defect in conidia formation. A significant difference was detected in osmotic, oxidative, or cell wall stress responses between the WT and ΔBsTup1 strains. Pathogenicity assays showed that virulence of the ΔBsTup1 mutant was dramatically decreased on wheat and barely leaves. Moreover, it was observed that hyphal tips of the mutants could not form appressorium-like structures on the inner epidermis of onion and barley coleoptile. Yeast two-hybrid assays indicated that BsTup1 could interact with the BsSsn6. RNAseq revealed significant transcriptional changes in the ΔBsTup1 mutant with 2369 genes down-regulated and 2962 genes up-regulated. In these genes, we found that a subset of genes involved in fungal growth, sporulation, cell wall integrity, osmotic stress, oxidation stress, and pathogenicity, which were misregulated in the ΔBsTup1 mutant. These data revealed that BsTup1 has multiple functions in fungal growth, development, stress response and pathogenesis in B. sorokiniana.

A novel effector, CsSp1, from Bipolaris sorokiniana, is essential for colonization in wheat and is also involved in triggering host immunity.

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host-pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N-terminal signal peptide, that is induced during early infection. A split-marker approach was used to knock out CsSP1 in the Lankao 9-3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana-infected wheat leaves, the salicylic acid-regulated genes TaPAL, TaPR1, and TaPR2 were down-regulated in the ∆Cssp1 strain compared with the wild-type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.

The FpPPR1 Gene Encodes a Pentatricopeptide Repeat Protein That Is Essential for Asexual Development, Sporulation, and Pathogenesis in Fusarium pseudograminearum.

Fusarium crown rot (FCR) and Fusarium head blight (FHB) are caused by Fusarium pseudograminearum and are newly emerging diseases of wheat in China. In this study, we characterized FpPPR1, a gene that encodes a protein with 12 pentatricopeptide repeat (PPR) motifs. The radial growth rate of the ΔFpppr1 deletion mutant was significantly slower than the wild type strain WZ-8A on potato dextrose agar plates and exhibited significantly smaller colonies with sector mutations. The aerial mycelium of the mutant was almost absent in culture tubes. The ΔFpppr1 mutant was able to produce spores, but spores of abnormal size and altered conidium septum shape were produced with a significant reduction in sporulation compared to wild type. ΔFpppr1 failed to cause disease on wheat coleoptiles and barley leaves using mycelia plugs or spore suspensions. The mutant phenotypes were successfully restored to the wild type levels in complemented strains. FpPpr1-GFP signals in spores and mycelia predominantly overlapped with Mito-tracker signals, which substantiated the mitochondria targeting signal prediction of FpPpr1. RNAseq revealed significant transcriptional changes in the ΔFpppr1 mutant with 1,367 genes down-regulated and 1,333 genes up-regulated. NAD-binding proteins, thioredoxin, 2Fe-2S iron-sulfur cluster binding domain proteins, and cytochrome P450 genes were significantly down-regulated in ΔFpppr1, implying the dysfunction of mitochondria-mediated reductase redox stress in the mutant. The mating type idiomorphic alleles MAT1-1-1, MAT1-1-2, and MAT1-1-3 in F. pseudograminearum were also down-regulated after deletion of FpPPR1 and validated by real-time quantitative PCR. Additionally, 21 genes encoding putative heterokaryon incompatibility proteins were down-regulated. The yellow pigmentation of the mutant was correlated with reduced expression of PKS12 cluster genes. Taken together, our findings on FpPpr1 indicate that this PPR protein has multiple functions in fungal asexual development, regulation of heterokaryon formation, mating-type, and pathogenesis in F. pseudograminearum.