Samples of roots were taken from the rhizosphere of robust potato plants. The gathered root samples were passed through а rigorous cleaning procedure. They were first washed with running tap water to get rid of debris, followed by washing three times with a sterile solution of distilled water. The root segments (less than 2 cm) were treated with 70% ethanol for 45 s to guarantee surface sterilization. After that, they were washed three times with a sterile solution of distilled water, dried on sterilized filter paper and inoculated on PDA. The plates were then exposed to incubation in the dark (25°C). Following incubation for seven days, fungal growth was noted, and separate colonies with various morphologies were chosen with the use of a sterile loop. After being carefully handled to avoid overgrowth, the colonies were grown on new PDA plates to produce pure cultures [16]. Phenotypic analysis of the isolate was done in the Plant Pathology Lab., Kafrelsheikh University.

An in-house fungal DNA isolation kit was used to extract genomic DNA from a 12-day-old culture, and a Denovix DS-11 spectrophotometer was used to assess the genomic DNA’s concentration and purity [20]. The procedures for polymerase chain reaction (PCR) and sequencing were followed [21]. Using BioEdit, the raw sequences used in this work were manually adapted and proofread. ClustalX was used to align the adjusted sequences [22]. Consensus sequences were established from the obtained sequences and added to Genbank after alignment. Molecular phylogenetic analysis was done using ver. 11.0.2 of MEGA-11 software [22]. Neighbor-joining analysis and information from whole nucleotide sequences were used to create a phylogenetic tree [22].

Barley kernel inoculum (BKI) from the endophytic fungus isolate (laboratory code ME23) was prepared as previously described by Elsharkawy et al. [16]. Colonized barley kernels (2 weeks after inoculation) were air dried and ground to 2 mm size then stored at 4°C until use. The Spunta potato cultivar, which has been verified free of viral infection using ELISA test, was gathered from the Agricultural Research Center’s brown rot research project (ARC, http://www.arc.sci.eg/default.aspx?lang=en) (accessed on 20 November 2024). The potato tubers were placed in 30 cm-wide pots that were filled with a specially made soil combination (1:2 w/w, sand: clay ratio, mixed with barley kernels colonized with the endophytic fungus at a final concentration 0.7% w/w). The potato virus Y subtype NTN and tobacco (Nicotiana tabacum) seeds were obtained from Plant Pathology Lab., Kafrelsheikh University, and the seeds were planted in 15-cm pots [23]. Smashed and extracted (in 0.1 M phosphate buffer solution, pH 7) tobacco leaves were used to produce viral inoculum for the following infection trials. The initial sap extract that had been purified was first filtered through cheesecloth. Potato leaves in both the main and tertiary stages were infected with a 10⁻¹ dilution of the infectious tobacco sap. In a greenhouse at 24°C with a 16-h photoperiod, inoculated tobacco and potato plants for PVY trials were maintained under insect-proof conditions.

Periodically, the foliage of infected potato plants was visually inspected to measure the evolution of the symptoms of the PVY disease over time. A consistent 0–4 grading system was used to grade the infection severity of each PVY-inoculated plant separately. The described symptom difference ranged from 0 = absence of obvious symptoms, 1 = moderate mottling of leaf mosaic (<20% infection area), 2 = noticeable mottling (20%–50% infected area), 3 = severe malformation, stunting, and mottling (>50% infected area), to the maximum score of 4 = severe mosaic, deformity, necrosis, and death of the plant. The following formula was used to determine the disease severity (DS) values at 7, 14, and 21 days post-viral infection (DPVI) [23]. The computation of the AUDPC (area under the disease progress curve) was done [24].

The analysis of proline content [25], carotenoids [26], and phenolic compounds were conducted [27,28]. For enzyme estimation in this experiment, the apical buds of the treated potato plants were the source. An enzyme estimation was performed using the clear supernatant obtained by centrifuging the sample (2 g) for 20 min at 2000 rpm and 2°C after it had been homogenized with 10 mL of phosphate buffer at pH 6.8. A UV spectrophotometer (Spectronic 20D, Thermo Electron) was used to detect the activity of the PPO (poly phenoloxidase), SOD (superoxide dismutase) and CAT (catalase) enzymes [23].

Seven weeks following the application treatment, the colonization of C. aleuritis in tobacco and potato roots was assessed. Ten randomly chosen plants’ roots were removed, and after being rinsed three times with sterile distilled water to get rid of any remaining soil, the roots were blotted dry and plated on PDA [16]. Colonies were enumerated from 100 root segments based on the isolates’ basic morphology.

Twelve weeks after planting, the treated potato plants’ yield characteristics were evaluated by harvesting. Each treatment group’s parameters, including the weight and number of tubers as well as the fresh and dry weights, were measured.

Leaves of potato and tobacco (treated and non-treated) plants were collected at 0, 2, 4 DAVI (days after virus inoculation) for tobacco and at 2 and 4 DAVI for potato. The methods of Elsharkawy et al. [23] and Livak and Schmittgen [29] were followed to carry out quantitative RT-PCR analysis for potato and RT-PCR for tobacco. The specific primers for tobacco and potato are listed in Tables 1 and 2 [30–33].

Four separate duplicates of each experiment were conducted. Post-hoc analysis of ANOVA results was applied. The means for each group of duplicates were used to illustrate the results. Using the Excel Tokei statistical software, the Least Significant Difference (LSD) at p < 0.05 was used to evaluate the statistical significance of differences between means.

PCR amplicons were utilized to identify the isolate. The studied strain has been preliminary identified as Coniothyrium aleuritis, but the exact taxonomic name will need to be classified through additional genetic studies. Through investigation, a 98.3% sequence identity match to C. aleuritis was found using BLAST comparisons and ITS gene sequencing. GenBank received the obtained ITS sequence and accepted it under accession OP862862.

Both tobacco and potato plants treated with C. aleuritis had considerably fewer disease symptoms than the untreated PVY-infected control group (Fig. 1). There was a noticeable and statistically significant decrease in PVY disease severity. The administration of C. aleuritis before PVY infection resulted in a significant decrease in disease severity. The relevant disease indices are presented in Fig. 2, which highlights the efficiency of C. aleuritis in reducing the severity of PVY infection in both potato and tobacco plants, confirming its potential as a protective strategy against the disease.

The ELISA findings showed that C. aleuritis plants had considerably less viral accumulation, whereas the non-treated group had the highest amount of viral accumulation (Fig. 3). Potato plants that were not treated had the highest degree of viral accumulation (0.43), whereas the C. aleuritis plants displayed much lower levels of viral accumulation (0.23). Interestingly, PVY concentration was significantly affected by applying C. aleuritis, falling from 0.49 to 0.18 in tobacco (Fig. 3). These findings highlight the multitude of C. aleuritis to mitigate the virus’s effects.

Treatments with C. aleuritis produced substantial impacts on fresh and dry weights of tobacco plants (Table 3). The comparison in tobacco plants demonstrated that the C. aleuritis-PVY treatment resulted in the maximum dry and fresh weights (1.1 and 10.1 g/plant, respectively). Table 4 shows the influence of C. aleuritis on major growth and yield measurements of potato. Based on the findings of the data analysis, it is apparent that all indicators have significantly improved. The C. aleuritis treatment produced the greatest fresh weight for PVY-infected potato (8.8 g/plant). C. aleuritis had a greater dry weight (0.91 g/plant) compared to PVY-infected group (0.42 g/plant). C. aleuritis-PVY infection treatment resulted in a modestly decreased fresh weight (8.8 g/plant) compared with the healthy group (10.8 g/plant). The C. aleuritis-PVY infection treatment had the greatest tuber weight (158 g/plant) among infected treatments. Similarly, the C. aleuritis-PVY infection treatment demonstrated the greatest tuber number (5.8) among infected treatments. Overall, the C. aleuritis-PVY treatment regularly displayed favorable impacts on growth indices, highlighting its ability to improve plant performance.

The treatment of potato plants with C. aleuritis before viral inoculation improves their PPO, CAT, and SOD activities (Table 5). Results indicate that C. aleuritis treatments increased accumulated PPO, CAT, and SOD by 0.14, 0.42 and 0.43 mol/gFW relative to the control group (0.06, 0.12, and 0.14 mol/gFW). Total phenols and proline contents in potato plants treated with C. aleuritis were 1.3 and 0.83 mg/g FW, respectively, compared with 0.98 and 0.64 mg/g FW in the control group. C. aleuritis-PVY infection treatment showed a greater amount of carotenoid (0.94 mg/g FW) than the infected control (0.63 mg/g FW) (Fig. 4).

According to our findings, 7 weeks after planting, potato and tobacco plants produced significant frequencies of C. aleuritis recording 92% and 95% for potato and tobacco plants, respectively (Fig. 5).

PR-1b and PAL1 genes were shown to express differently in C. aleuritis potato plants compared to healthy and infected control (Fig. 6). In comparison to the control, the potato plants treated with C. aleuritis had relative expression levels of PR-1b that were 3 and 5-folds higher at 2 and 4 days after PVY inoculation (DAVI), respectively. PAL1 showed upregulation of expression in all treatments, especially treatment with C. aleuritis (2.1-fold) and (3.5-fold) at 2 and 4 (DAVI) compared to the control expression levels (Fig. 6).

Transcription levels of PR1 and Coi1 genes associated with pathogenesis in tobacco are shown in Fig. 7. In C. aleuritis, the transcription of the PR1 gene in tobacco plants started to be induced one day after the induction treatments and continued at high levels for four days after the inoculation. Coi1 transcription initially appeared in the C. aleuritis treatment at 1 day after infection and remained elevated 4 days later.