Soft rot of olive drupes caused by Phytophthora oleae is an emerging disease first reported in Italy. In this study, RNA-seq analysis was used to investigate the transcriptional responses of olive drupes to P. oleae. Two biological pre-treatments were also evaluated, consisting of the culture filtrate of the antagonistic filamentous fungus Trichoderma atroviride and a cell suspension of the antagonistic yeast Candida oleophila. Both treatments were applied 24 h before pathogen inoculation. Although complete protection was not achieved, both pre-treatments significantly reduced rot severity in inoculated drupes. Severity of Phytophthora rot was assessed at 24, 72, and 168 hours post inoculation (hpi) using an empirical rating scale. Both biological pre-treatments significantly reduced rot severity, with comparable reductions of 56% for T. atroviride culture filtrate and 52% for C. oleophila cells. At 72 hpi, clear differences in lesion size were observed, and this time point was therefore selected for RNA-seq analysis. A total of 2,307, 1,714, and 1,816 genes were differentially expressed in drupes inoculated with P. oleae alone, pre-treated with T. atroviride culture filtrate and subsequently inoculated with P. oleae, or pre-treated with C. oleophila and subsequently inoculated with P. oleae, respectively, compared with wounded control drupes. Selected RNA-seq-responsive genes were further validated by RT-qPCR at 24, 72, and 168 hpi. The combined RNA-seq and RT-qPCR results indicate that both biological pre-treatments were associated with marked modulation of defense- and stress-related transcriptional responses in olive drupes, while also revealing partially distinct regulatory signatures during P. oleae infection.
Bacterial stalk rot (BSR), caused by Dickeya zeae, is an emerging threat to maize production worldwide. However, information on the genetic diversity of Indian D. zeae populations and reliable molecular detection methods remains limited. In this study, seventeen D. zeae isolates collected from four major maize-growing regions of India (Telangana, Delhi, Himachal Pradesh, and Uttarakhand) were characterized using repetitive sequence-based PCR (rep-PCR) with REP, ERIC, and BOX primers. UPGMA cluster analysis revealed a high level of genetic heterogeneity, with genetic clustering largely independent of geographic origin, indicating extensive diversity within the Indian D. zeae population. Species-specific primers targeting the recN and fliC genes were developed, producing amplicons of 198 and 230 bp, respectively. The fliC primer pair showed higher analytical sensitivity, detecting as little as 0.00001 ng μL-1 of genomic DNA, compared with 0.0001 ng μL-1 for the recN primer pair. Both primer pairs specifically detected D. zeae without cross-reactivity with non-target bacterial species. Validation of the BIO-PCR assay using 96 field-derived maize samples demonstrated that the fliC-based assay detected D. zeae in 100% of symptomatic and 15.38% of asymptomatic samples, outperforming the recN-based assay (96.77% and 9.23%, respectively). This study represents the first comprehensive assessment of the genetic diversity of Indian D. zeae populations and the first report from India describing a recN- and fliC-based BIO-PCR assay for the sensitive and specific detection of D. zeae in maize.
Rice false smut, caused by the pathogen Ustilaginoidea virens, significantly reduces both the yield and quality of rice crops. Effective management of this pathogen is challenging due to the distinctive way U. virens infects plants. Consequently, there is a need for techniques that allow for early detection and monitoring to limit the spread of disease. Loop-mediated isothermal amplification coupled with a lateral flow assay overcomes the drawbacks of existing U. virens detection methods, which are often slow, more expensive, dependent on sophisticated laboratory equipment, and prone to false-positive results. In this study, the Uvβ1 gene, unique to U. virens, was selected as the target for assay development. The modified primers we developed successfully identified U. virens DNA without cross-reacting with closely related pathogens. Under optimized reaction conditions, the assay exhibited high sensitivity, detecting as little as 100 fg of U. virens DNA. Moreover, by using buffers to extract crude pathogen DNA, the LAMP-LFA facilitated the identification of U. virens in rice grains in under 64 minutes, eliminating the need for specialized equipment. Consequently, it demonstrates that the LAMP-LFA is a sensitive, rapid, and user-friendly diagnostic tool with strong potential for field surveillance, early disease diagnosis, and effective management of disease outbreaks, thereby helping to minimize yield losses.
Anthracnose fruit rot of blueberry, caused by Colletotrichum spp., is a devastating post-harvest rot that threatens production systems. While the defense response of blueberries to infection has been examined, characterization of the pathogen’s biology has been limited primarily to microscopy. This study evaluated the infection process of Colletotrichum fioriniae during infection of blueberry fruits of the susceptible cultivar ‘Jersey’. Fruits were inoculated with a conidial suspension of C. fioriniae. RNA was extracted and sequenced after 1-, 12-, 24-, 48-, 72-, 96-, 120-, and 168-hours post-inoculation (hpi) as well as 14 days post-inoculation (dpi). Sequenced reads were aligned to a reference C. fioriniae genome and a de-novo transcriptome assembled to identify novel transcripts, which were then annotated. Analysis of differentially expressed genes (DEGs) was performed to examine trends in expression with a focus on genes relating to pathogenic processes. This analysis identified major transitions in expression occurring at 12 hpi, between 24 and 72 hpi, at 96 hpi, 120 hpi, and again at 14 dpi, providing molecular context to the infection stages previously characterized. Examining expression patterns of carbohydrate active enzymes (CAZymes) demonstrated extensive upregulation of plant-cell-wall degrading enzymes (PCWDEs) from 48 to 96 hpi. Examination of DEGs identified extensive transcriptional activity related to the production of the polyketides aurofusarin and depudecin at 96 and 120 hpi respectively, suggesting previously unreported roles of these polyketides in the infection process. Our results expand understanding of the blueberry–C. fioriniae interaction on a molecular level and validate previous microscopic characterization of the pathosystem.
Wheat productivity is severely constrained by spot blotch caused by Bipolaris sorokiniana, with disease severity further exacerbated by intraspecific competition for resources under dense planting conditions. This study explores a sustainable, biologically driven strategy to enhance wheat tolerance to pathogen stress under varying plant densities. We demonstrated that plant growth-promoting rhizobacteria (PGPR)-mediated defense priming enhances wheat resilience against B. sorokiniana across different levels of competition. Increasing plant density led to a general reduction in growth traits in both primed and non-primed plants; however, primed plants consistently exhibited superior morphological performance compared to their non-primed counterparts. Under pathogen pressure, PGPR-primed plants showed enhanced tolerance across all density regimes, with protection levels remaining stable despite increasing competition intensity. Biochemical analyses, including photosynthetic pigments, oxidative stress markers, enzymatic as well as non-enzymatic antioxidants, and defense-related enzymes revealed that enhanced defense responses in primed plants were specifically activated upon pathogen exposure, confirming the establishment of a true priming state. At the molecular level, epigenetic analysis of the 2 kb upstream regulatory regions of PR1 and PR3 genes at low density (D1) revealed differential DNA methylation patterns, as assessed by MspI/HpaII restriction digestion, indicating priming-associated epigenetic reprogramming. These changes are likely linked to enhanced transcriptional activation of defense-related genes upon pathogen challenge. In contrast, non-primed plants at the highest density (D3) exhibited the greatest susceptibility under combined competition and disease stress. Bacillus amyloliquefaciens-mediated priming mitigated the fitness costs associated with high-density stress while reducing disease severity. Notably, PGPR-primed plants maintained improved yield performance across all planting densities. Collectively, these findings establish PGPR-induced defense priming as an eco-friendly and effective strategy to enhance wheat resistance to B. sorokiniana and sustain productivity under dense planting conditions.
Atractylodes macrocephala (A. macrocephala) is a medicinal plant that is highly susceptible to root rot caused by Sclerotium rolfsii (S. rolfsii). Polysaccharides derived from the cell wall of S. rolfsii (SR) have been reported to act as elicitors of immune responses in A. macrocephala; however, the upstream molecular components associated with SR-induced immune responses remain poorly characterized. In this study, three candidate LysM receptor genes responsive to SR treatment, AmLYK4, AmLYK5, and AmCEBiP, were identified and subjected to functional characterization. Sequence and domain analyses indicated that AmLYK4 and AmLYK5 encode LysM receptor-like kinases, whereas AmCEBiP encodes a LysM receptor-like protein lacking an intracellular kinase domain. SR treatment resulted in rapid and transient induction of AmLYK4, AmLYK5, and AmCEBiP transcripts. Bimolecular fluorescence complementation (BIFC) assays in Nicotiana benthamiana indicated that the three candidate proteins could occur in close spatial proximity under the assay conditions, with stronger fluorescence observed following SR treatment; however, this difference did not establish enhanced complex formation. In A. macrocephala leaves, transient overexpression of each candidate gene was associated with enhanced expression of SR-induced defence-related genes, whereas virus-induced gene silencing attenuated these responses. Collectively, these findings indicate that AmLYK4, AmLYK5, and AmCEBiP are associated with SR-induced immune responses and may contribute to glycan-responsive defence regulation in A. macrocephala. Direct binding between these candidate proteins and SR polysaccharides remains to be established.
Fusarium graminearum, a major causal agent of Fusarium head blight, poses a persistent threat to cereal production and food safety, necessitating the development of sustainable biocontrol strategies as alternatives to chemical fungicides. In the present study, a lactic acid bacterium, Lacticaseibacillus paracasei MYSN17, obtained from the repository of Applied Mycology Laboratory was evaluated for its antifungal activity against F. graminearum MTCC 1893 and the molecular basis underlying its antagonistic activity. MYSN17 demonstrated potent antifungal activity in agar overlay and dual-culture assays, resulting in significant suppression of fungal growth. The cell-free supernatant (CFS) displayed a concentration-dependent inhibition of fungal growth at 20%, resulting in 88.33% inhibition. Scanning electron microscopy revealed morphological alterations on treatment with MYSN17 and its CFS. Further, CFS treatment effectively prevented F. graminearum colonization in maize kernels under storage conditions. LC-MS/MS profiling identified eleven organic acids in the CFS, suggesting a metabolite-mediated mechanism of fungal inhibition. Genome mining revealed putative biosynthetic potential encoding bacteriocins, lantipeptides, non-ribosomal peptide synthetases (NRPS), terpenes, and type III polyketides. Collectively, the findings establish L. paracasei MYSN17 as an eco-friendly biocontrol agent for food preservation and sustainable management of F. graminearum.
Circular leaf spot (CLS) disease, primarily caused by the fungal pathogen Colletotrichum siamense, infects rubber trees and has emerged as a significant threat to the primary source of natural rubber. Understanding the candidate genes of clonal resistance to this disease is crucial for ensuring stable rubber production. Here, comparative transcriptomic analysis via RNA-sequencing provides an informative dataset of differentially expressed genes for the defense responses of two rubber tree clones, susceptible RRIM600 and resistant RRIT3904, after infection with C. siamense for one week. The results revealed a growth-defense trade-off in the RRIT3904 clone involving the downregulation of photosynthesis-related genes and activation of amino acid metabolism, suggesting metabolic remodeling for immune signaling and defense-related precursor synthesis in host resistance. Conversely, the susceptible clone RRIM600 exhibited upregulation of photosynthesis genes and a decline in secondary metabolite biosynthesis, which potentially reflects metabolic hijacking of C. siamense in this clone. Key upregulated genes in RRIT3904 include SPX-domain-containing proteins, suggesting balanced phosphate homeostasis in host resistance. Differentially expressed genes between clones that were mock-inoculated versus C. siamense-infected were also analyzed. Together, these results highlight critical pathways that distinguish resilience from susceptibility, offering a molecular basis for breeding programs aimed at mitigating CLS disease in rubber tree plantations.
The plant-associated endophytic fungi are a source of biologically active metabolites, which have not only agricultural importance but also biotechnological potential. A total of 81 fungal endophytic isolates belonging to 76 fungal species were isolated from Heteropogon contortus in the present study. Of these species, Aspergillus ochraceus, Chaetomium subaffine, Penicillium commune, Penicillium islandicum and Pestalotiopsis guepinii were chosen for further studies. The phytochemical screening showed the presence of flavonoids, phenolics, glycosides, alkaloids, steroids, and triterpenoids, and C. subaffine had the widest spectrum. The methanolic extract of H. contortus and the ethyl acetate extract of C. subaffine, have the highest redox potential in cyclic voltammetry analysis. Regarding the growth-promoting effect on plants, 100% germination was observed for both chili and maize, with the highest growth response in C. subaffine extracts. Using the Orbitrap high-resolution LC–MS, 10 major metabolites were identified in the host extract, while 5 metabolites were identified in C. subaffine. The molecular docking results showed that the compound (+)-aphidicolin exhibited good binding affinity to various proteins, including 2CDU (−11.380 kcal mol-1), 6KU3 (−11.283 kcal mol-1) and 3OGM protein complex (−13.283 kcal mol−1), whereas the compound andrographolide exhibited high binding affinity to 6KU3 (−13.283 kcal mol−1) and 4LDY (−8.965 kcal mol−1). The results of this simulation analysis, done in Normal Mode (NM) version, also showed that (+)-aphidicolin–2CDU and artemisinin–2CDU remain stable. The present results indicate H. contortus and its endophytic fungi as potential sources of antioxidant and growth promoting metabolites for sustainable agriculture.
Septoria nodorum blotch (SNB) is an economically important fungal disease of wheat caused by Parastagonospora nodorum. It is primarily controlled by the breeding of resistant wheat cultivars, but experience over the last 50 years shows that new pathogen populations soon evolve that are more virulent on the current popular cultivars. In this study, we assembled a panel of 360 P. nodorum isolates. The collection resolved into eight subpopulations. One core and seven transient populations were found possessing contrasting characters in term of spatial and temporal distribution, mating-type, effector haplotypes and patterns of intact and degraded copies of a Tc-1 mariner transposon, called Molly. Molly can proliferate and randomly insert throughout the fungal genome. Its multiplication in sexual population likely triggered RIP which partially explains the extensive genetic diversity. Molly activities, together with sexual reproduction and different selection pressures, are all part of evolutionary forces to shape the Australian P. nodorum population structure and explain a model where new lineages of this important pathogen of wheat could be formed. When tested on wheat, the recently emerged groups exhibited greater pathogenicity on modern elite cultivars consistent with the low-amplitude boom-and-bust cycle observed previously. This study identified and characterised potential mechanisms which may lead to the pathogen’s better adaptation and host specialisation. The study also suggests practical measures to improve the efficiency and longevity of resistance breeding for SNB.
