Seedlings of sugarcane cultivar ROC22 were grown in a controlled greenhouse at 25°C ± 2°C, 60%–70% relative humidity (RH), and a 12-hour light/12-hour dark photoperiod (≈200 μmol m⁻² s⁻¹). These same conditions were used for collecting diseased material and for all downstream experiments to ensure comparability. Healthy ROC22 plants maintained under identical conditions served as inoculation recipients and mock controls.

Genomic DNA was extracted from fresh mycelium (≈3–5 days on PDA) using the cetyltrimethylammonium bromide (CTAB) (Doyle and Doyle, 1987) method. The ITS region was amplified with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990). PCR conditions were as follows: 95°C for 3 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s; final extension at 72°C for 5 min. Amplicons were verified on a 1%–1.5% agarose gel and Sanger-sequenced bidirectionally.

Consensus ITS sequences were queried against the NCBI nr/nt databases using BLAST to confirm identity (Altschul, 1997). BLASTn of the ITS sequence showed ≥99% identity to N. oryzae (reference GenBank accession KU254608.1). Sequences from this study will be deposited in GenBank upon acceptance. Sequences were aligned with MAFFT v7 (Katoh and Standley, 2013). ModelFinder in IQ-TREE selected the best-fit substitution model (Kalyaanamoorthy et al., 2017). Maximum-likelihood trees were inferred with IQ-TREE 2 using 1,000 ultrafast bootstrap replicates (Minh et al., 2020). Trees were rooted with Arthrinium arundinis (Apiosporaceae), and node support ≥95 was considered strong.

Mycelial plugs and spore suspensions were used to assess pathogenicity in both in vitro (detached leaves) and in vivo (attached leaves) settings. Just before inoculation, leaves were lightly punctured with a sterile needle for wounding treatments; 5-mm PDA plugs with growing mycelium were applied to the leaf surface in mycelial-plug assays; sterile PDA plugs were used as controls. The inoculum was evenly applied to the specified spot in spore-suspension experiments, while controls were given sterile water. Symptoms were noted at 12, 24, 36, 48, 60, and 72 hours after inoculation, and the plants were maintained in a climate chamber (PRX-450A, Ningbo Saifu Laboratory Equipment, Ningbo, Zhejiang, China) under carefully monitored conditions.

Leaf tissues were taken every 24 hours for 6 days post inoculation (0–6 dpi) to measure MDA, a marker of lipid peroxidation, and to assess the activity of SOD, POD, and PAL. Three biological replicates were used for each treatment and time point in all experiments; technical replicates were averaged before statistical analysis. Commercial colorimetric kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; cat. nos.: SOD, A001-3-1; POD, A084-3-1; PAL, A137-1-1; and MDA, A003-1-2) were used to test SOD, POD, PAL, and MDA according to the manufacturer’s instructions. MDA is reported as nmol g⁻¹ fresh weight (FW), while enzyme activity is expressed as U mg⁻¹ protein. Total chlorophyll (a + b) was measured spectrophotometrically at 663 and 645 nm from 80% (v/v) acetone extracts using a UV–Vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan) with 1-cm quartz cuvettes; concentrations were calculated per Lichtenthaler (1987) and expressed as mg g⁻¹ FW. Enzyme activity data were analyzed using a two-way ANOVA with treatment and time (0–6 dpi) as fixed factors and their interaction, implemented in R using the drc package (Ritz et al., 2015).

Eight fungicides commonly used in sugarcane—chlorothalonil, difenoconazole (10%), copper hydroxide, pyraclostrobin + metiram (60%), carbendazim, metalaxyl + hymexazol, myclobutanil, and zhongshengmycin—were evaluated against N. oryzae. For the in vitro assay, PDA was amended with each fungicide at 50, 25, 12.5, and 6.25 μg mL⁻¹. Plates were inoculated with N. oryzae and incubated. Mycelial growth inhibition by fungicides was assessed using colony area rather than diameter to obtain more accurate estimates of radial expansion. Colony radius (r) was measured in two perpendicular directions, and colony area (A) was calculated as A = πr². Colony growth was quantified from colony area and computed from measured diameter: A = π(D/2)². For each fungicide, the inhibition rate (%) was calculated relative to untreated controls, as follows:

Dose–response data were fitted using a log-dose probit regression model to obtain toxicity regression equations, correlation coefficients, and EC₅₀ values. Regression curves with 95% confidence intervals were generated to visualize fungitoxicity trends.

For the plant assay, detached-leaf or intact-plant tests were conducted under the same greenhouse conditions using a formulation of 60% pyraclostrobin + metiram and 10% difenoconazole and applied at labeled rates. Disease severity or mycelial growth inhibition was recorded, and treatments were compared statistically at α = 0.05.

Data were analyzed using SPSS v18.0, and figures were prepared using GraphPad Prism v8.0. Differences among treatments were assessed using one-way ANOVA. When significant differences were detected, means were separated using Tukey’s test at p < 0.05. In figures, values are presented as means ± standard error (SE), and groups that differ statistically are indicated by different letters. Dose–response data were fitted with a four-parameter log-logistic (LL.4) model using the drc package in R (Ritz et al., 2015) to estimate EC₅₀ and 95% confidence intervals.

A fungal isolate was consistently recovered from symptomatic sugarcane leaves (Figures 1A–C). Colonies on PDA were initially white and later darkened to gray-black with dense aerial mycelia (Figures 2A, B). Conidia were black, spherical to slightly ellipsoid, and measured approximately 12–15 μm in diameter (Figures 2C, D). These characteristics are consistent with Nigrospora species.

Gel electrophoresis of the PCR-amplified ITS product revealed a specific fragment of approximately 500 bp, confirming successful amplification. The purified PCR product was sequenced, and the resulting sequence was analyzed using BLAST on the NCBI website (www.ncbi.nlm.nih.gov). Our ITS sequence matched N. oryzae with ≥99% identity (reference: KU254608.1). In accordance with the morphological observations, the isolate clustered within the N. oryzae clade in a phylogenetic tree constructed in MEGA v5.0 using the neighbor-joining method with 1,000 bootstrap replicates (Figure 3). Based on these combined results, the pathogen was identified as N. oryzae. The associated disease in sugarcane was named as Nigrospora leaf spot, following nomenclature guidelines for plant fungal diseases (Zheng, 2012).

Necrotic lesions developed consistently only on mechanically wounded leaves inoculated with either spore suspensions or mycelial plugs of N. oryzae. No symptoms were observed on unwounded inoculated leaves or on any of the control treatments during the 72-hour observation period. Wounded leaves inoculated with the pathogen exhibited visible necrotic lesions within 3–4 days (Figures 4A–C), whereas unwounded leaves remained symptomless (Figures 4B, D). The pathogen was successfully re-isolated from symptomatic tissue, thereby fulfilling Koch’s postulates and confirming its pathogenicity.

Mechanical wounding was essential for successful infection by N. oryzae. Wounded leaves inoculated with mycelial plugs developed necrotic lesions within 24 hours, which enlarged progressively over the next 72 hours (Figure 4A). Detached leaves inoculated with mycelial plugs exhibited even faster symptom onset (24–36 hours), indicating greater susceptibility of excised tissue compared with intact plants (Figure 4B). Inoculation with spore suspensions produced lesions only on wounded leaves, with visible necrosis appearing at 48–60 hours and becoming more pronounced by 72 hours (Figure 4C). Throughout the 72-hour observation period, unwounded leaves inoculated with spore suspensions remained symptomless (Figure 4D). The pathogen was successfully re-isolated from symptomatic tissues, completing Koch’s postulates. Infection significantly reduced chlorophyll a, chlorophyll b, and total chlorophyll in wounded inoculated leaves compared with controls (Table 1). No significant changes were observed in unwounded or mock-inoculated leaves.

In vitro pathogenicity tests further demonstrated that N. oryzae rapidly initiates lesion formation on wounded sugarcane leaves. When mycelial plugs were applied to injured tissue, faint reddish lesions developed within 24–36 hours and expanded progressively, with visible mycelial proliferation by 72 hours (Figure 5A). Detached leaves inoculated with mycelial plugs showed similar symptoms, including red patches and central yellowing by 48 hours (Figure 5B). Spore suspension inoculation also resulted in disease development, but with a slightly slower onset. Mild reddening at wound sites appeared at approximately 36 hours, followed by the enlargement of lesions with reddish-brown margins and mycelial development by 72 hours (Figure 5C). Throughout the 72-hour observation period, unwounded leaves inoculated with spore suspensions remained symptomless, resembling the uninoculated controls (Figure 5D). All control treatments (including sterile PDA plugs and sterile water) failed to produce lesions, suggesting that symptom development was pathogen-specific.

Temperature significantly influenced radial mycelial growth on PDA (Figure 6A). The isolate exhibited maximal colony expansion at 25°C–30°C. Growth declined markedly at 20°C and 35°C, and no growth occurred at 40°C. Differences among temperatures were statistically significant (p < 0.05). Similarly, pH strongly affected mycelial development (Figure 6B). The fastest growth was recorded at pH 7, with moderate growth at pH 5–6 and pH 8. Mycelial growth was substantially reduced under more acidic (pH 4) or alkaline (pH 9) conditions.

Infection by N. oryzae significantly increased the activity of antioxidant enzymes in sugarcane seedlings. The activities of SOD and POD were markedly higher in infected seedlings than in controls across multiple time points (ANOVA, p < 0.05; Figure 7). Similarly, PAL activity and MDA content were significantly elevated in infected seedlings compared with controls (ANOVA, p < 0.05; Figure 8), indicating enhanced oxidative stress and activation of defense-related biochemical pathways.

Fungicide sensitivity assays revealed a clear hierarchy in the inhibitory activity of the eight tested compounds against N. oryzae. Among them, 60% pyraclostrobin + metiram was by far the most potent, achieving 95.24% inhibition at the test concentration and exhibiting the lowest EC₅₀ value (0.01 μg mL⁻¹). Its performance was significantly superior to all other treatments (ANOVA, p < 0.05; Tables 1, 2), identifying it as the most effective candidate for disease management; 10% difenoconazole ranked second, providing 83.05% inhibition at 50 μg mL⁻¹ with an EC₅₀ of 9.698 μg mL⁻¹. Although its efficacy declined more rapidly at lower concentrations compared with pyraclostrobin + metiram, it still demonstrated strong and consistent antifungal activities. A moderate level of control was achieved with 75% chlorothalonil, which produced 53.17% inhibition at the highest tested concentration and an EC₅₀ of 50.149 μg mL⁻¹, suggesting that only relatively high doses would be effective.

In contrast, 25% myclobutanil showed limited activity, reaching only 39.49% inhibition at 50 μg mL⁻¹ and presenting a high EC₅₀ of 350.727 μg mL⁻¹. The remaining fungicides—50% carbendazim, 3% metalaxyl + hymexazol, 3% zhongshengmycin, and especially 46% copper hydroxide—exhibited poor to negligible inhibition, with copper hydroxide being virtually ineffective (4.70% inhibition; EC₅₀ = 8,817.992 μg mL⁻¹). Collectively, these results indicate that only a subset of the tested fungicides, particularly pyraclostrobin + metiram and difenoconazole, possess strong inhibitory potential against N. oryzae.

This study provides the first integrated characterization of N. oryzae associated with sugarcane leaf spot and clarifies key aspects of its infection biology, pathogenicity, host physiological responses, and fungicide sensitivity. Although the current incidence and economic impact of this disease under natural field conditions have not yet been quantified, observations made during sample collection indicated that leaf spot symptoms were sporadic and strongly associated with plants showing visible mechanical damage or stress. Our findings demonstrate that N. oryzae acts as a wound-dependent opportunistic pathogen in sugarcane, consistent with previous reports in other hosts where infection typically requires mechanical injury or weakened tissue (Liu et al., 2021; Chen, 2020). The absence of symptoms on intact leaves confirms that the pathogen cannot penetrate healthy epidermal tissue, underscoring the role of physical damage such as insect feeding, hail injury, and harvest-related wounds as critical infection courts. This wound dependency provides a plausible explanation for the irregular and localized nature of disease occurrence in the field, where infection is likely limited to areas experiencing frequent tissue injury rather than widespread epidemic development. The lesions observed on wounded leaves, characterized by necrotic spots with darkened centers, closely align with symptom descriptions for cotton rose (Wang et al., 2022), ginger (Han et al., 2021), and wild rice (Lu et al., 2023). This suggests that N. oryzae employs a comparable necrotrophic strategy across hosts. The rapid lesion expansion observed within several days of inoculation further supports its opportunistic lifestyle and highlights its potential threat under field conditions, where wounds are common.

Inoculated plants exhibited marked reductions in total chlorophyll, indicating impaired photosynthetic capacity. Similar chlorophyll loss has been documented in ginger and barley infected by N. oryzae and related necrotrophic fungi (Liu et al., 2021; Geetha et al., 2005). Elevated MDA levels in infected leaves suggest enhanced lipid peroxidation and membrane damage, which are typical markers of oxidative stress during pathogen challenge (Song et al., 2022). The significant increases in SOD and POD activities indicate the activation of the antioxidant system as the plant attempts to detoxify reactive oxygen species (ROS). Enhanced PAL activity further suggests the stimulation of the phenylpropanoid pathway, which contributes to the lignification and synthesis of antimicrobial phenolics (Wang et al., 2024). Together, these responses reflect a typical host reaction to necrotrophic pathogens, which often provoke intense oxidative bursts that contribute to cell death and lesion formation. In the context of field infections, such wound-induced oxidative responses may inadvertently facilitate pathogen colonization by accelerating localized cell death at injury sites, thereby favoring necrotrophic establishment. N. oryzae exhibited optimal mycelial growth at 25°C–30°C and pH 7, which is consistent with reports from other hosts and environmental isolates (Widmer et al., 2006; Liu et al., 2021). These conditions closely align with the climate of many sugarcane-growing regions, particularly in subtropical areas, suggesting that the pathogen can readily proliferate in the field when temperatures are warm and humidity is high.

Fungicide screening revealed substantial variability in N. oryzae’s sensitivity to different active ingredients, and colony area-based inhibition showed greater differentiation among chemistries than traditional diameter measurements. The mixture of pyraclostrobin and metiram exhibited the highest inhibitory activity, achieving >90% inhibition across all concentrations and an exceptionally low EC₅₀ (~0.01 μg mL⁻¹). This result reflects the rapid shutdown of mitochondrial respiration by strobilurins (Fungicide Resistance Action Committee (FRAC) 11) (Table 3), which block electron transfer at the Qo site of Complex III, combined with the protective action of metiram. Such potency suggests that this formulation is particularly effective for the rapid suppression of early infection and sporulation, aligning with prior reports of strong quinone outside inhibitor (QoI) performance against Nigrospora species in other crops (Massi et al., 2021; Yin et al., 2023). Difenoconazole (FRAC 3) ranked second in efficacy (EC₅₀ ≈ 1 μg mL⁻¹). Its inhibition of ergosterol biosynthesis, which limits hyphal elongation and membrane formation, is consistent with its broad-spectrum activity against many ascomycetes. Given its systemic properties and reliable performance, difenoconazole is likely to be effective as both a preventive and early curative option against N. oryzae. Chlorothalonil showed moderate inhibitory activity (EC₅₀ ≈ 13 μg mL⁻¹). As a multi-site protectant (FRAC M05) that disrupts protein thiol groups, its moderate performance agrees with earlier observations that Nigrospora isolates often show partial tolerance to multi-site fungicides. Nonetheless, chlorothalonil may still contribute to resistance management when used in alternation with more potent chemistries. In contrast, copper hydroxide, myclobutanil, metalaxyl + hymexazol, carbendazim, and zhongshengmycin exhibited poor inhibition, yielding EC₅₀ values exceeding 350–10,000 μg mL⁻¹. These concentrations are far above field-applicable rates, indicating intrinsic or acquired tolerance. Several mechanistic explanations align with this observation: metalaxyl targets oomycetes rather than ascomycetes, carbendazim resistance is widespread globally, and copper-based formulations primarily target bacterial pathogens rather than filamentous fungi. Similar poor sensitivity to copper compounds has been reported in other Nigrospora isolates (Hawkins, 2024).

Taken together, the EC₅₀ values, inhibition profiles, and FRAC mode-of-action classifications clearly identify pyraclostrobin + metiram and difenoconazole as the only highly effective fungicidal options for N. oryzae. However, because QoI resistance evolves rapidly in many plant pathogenic fungi, they should not be used as single-dependency chemistries. Instead, integrated disease management should combine targeted fungicide selection with cultural practices that reduce wounding, including minimizing mechanical injury, managing insect pests that create entry points, and avoiding unnecessary handling damage. Given the wound-inducible nature of N. oryzae, disease development under field conditions is likely contingent upon the frequency and severity of physical damage rather than aggressive host penetration, emphasizing the importance of injury prevention as a primary disease control strategy. As N. oryzae infection is wound-dependent, reducing physical injury may significantly limit disease onset. When combined with rational fungicide use and rotation across FRAC groups, these strategies provide a robust approach for managing N. oryzae leaf spot.