Identification, characterization, and sensitivity to phytochemicals of a novel Curvularia species associated with leaf spot disease on Curcuma kwangsiensis

Abstract

A leaf spot disease affecting Curcuma kwangsiensis (Zingiberaceae) has been observed in Qinzhou City, Guangxi Province. Infected leaves exhibit yellow-brown spots that progressively expand and eventually lead to leaf death. Curvularia isolates were obtained from the diseased leaves with tissue isolation and single spore purification methods. To accurately identify these isolates, we analyzed their morphological characteristics and phylogenetic relationships using combinations of ITS, GAPDH, and EF-1α gene sequences. Phylogenetic analysis showed that the investigated strains formed a distinct clade separate from other recognized Curvularia species. Furthermore, the strains exhibited differences in conidiophore size and conidia shape/size. Based on phylogenetic studies, morphology, and pathogenicity tests, the pathogen was identified as a new species named Curvularia qinzhouensis. Optimal conditions for mycelial growth were observed at 30 °C and pH 8. The sensitivity of the pathogen to various phytochemicals was also examined. Honokiol, thymol, and citral demonstrated effective antifungal effects, with EC₅₀ values of 6.72 ± 1.75, 25.74 ± 4.30, and 54.24 ± 4.69 µg/ml, respectively. The present investigation provides the first report of leaf spot disease on C. kwangsiensis caused by C. qinzhouensis, and valuable insights for the prevention and control of this disease.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-77524-1.

Introduction

Curcuma kwangsiensis S. G. Lee et C. F. Liang is a perennial herbaceous plant belonging to the Zingiberaceae family, commonly grown in the southwestern region of China. It holds significant medicinal value in traditional medicine, with recent studies confirming its efficacy in liver protection, tumor inhibition, antioxidant activity, blood lipid reduction, and immune system modulation¹. Apart from its medicinal properties, its attractive thin green leaves and vibrant cylindrical flower clusters make it a popular choice for ornamental purposes in flower beds². However, with the increasing demand and expansion of cultivation areas, various diseases have emerged, posing a threat to yield and quality. For example, Epicoccum latusicollum has been identified as a pathogen causing leaf blight and defoliation in C. kwangsiensis³. Nonetheless, many pathogens responsible for diseases in C. kwangsiensis remain unidentified.

Curvularia encompassing a diverse range of species including plant, animal, and human pathogenic fungi, is a genus belonging to the Pleosporalean order⁴. The distinguishing feature of Curvularia is the formation of brown distoseptate conidia, typically with lighter end cells and enlarged middle cells. However, due to morphological similarities, the identification of Curvularia species cannot be accurately achieved solely through morphological features and analyses of the ITS sequence^5,6. Consequently, the investigation of species diversity in Curvularia has been conducted using multi-locus sequence analysis. Scientists have employed multi-locus sequence analysis, utilizing the internal transcribed spacer (ITS) region of rDNA, as well as the protein-coding loci glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-a (EF-1α), to investigate species diversity in Curvularia and its phylogenetic relationships with related genera^7,8. To date, approximately 130 species have been recognized within the genus Curvularia based on DNA sequences^4,9.

Certain species of Curvularia are responsible for leaf spot in various crops, such as C. asianensis and C. eragrostidis in Sansevieria trifasciata, while C. lunata is a pathogen of sunflower (Helianthus annuus)^10,11. Effective management of the leaf spot disease in C. kwangsiensis requires identification of the pathogen and implementation of control measures. In addition, determining the optimal growth temperature and pH for these pathogenic fungi is crucial for understanding their ecological characteristics, developing control strategies, preventing disease outbreaks, and ensuring the successful cultivation of C. kwangsiensis. One area of current research focuses on the use of phytochemicals to manage plant diseases. For instance, carvacrol has shown inhibitory effects on the mycelial growth of three leaf pathogens (Xanthomonas perforans, Alternaria tomatophila, and Podosphaera xanthii)¹². Similarly, thymol exhibits strong antifungal properties against Fusarium graminearum by damaging the cell membrane through lipid peroxidation¹³. The objective of this study was to identify the causative agent of leaf spot disease in C. kwangsiensis and investigate potential phytochemical compounds for controlling the potentially economically important agricultural pathogen.

Results

Sampling, fungal isolation and pathogenicity assay

Curcuma kwangsiensi, cultivated in plantations (∼1.3 ha) in Qinzhou City, Guangxi Province (21° 51’ 00′′ N, 108° 44’ 00′′ E), experienced an outbreak of leaf spot disease in May 2021. Approximately 15% (n = 200) of the plants were infected. Initial symptoms included yellow, watery, irregular lesions on young leaves, which progressed to affect entire tender leaves. Eventually, the leaves turned yellow, withered, and led to plant death (Fig. 1). In this study, twenty-six isolates resembling the genus Curvularia were obtained from symptomatic plants. Three representative isolates (CK43.1, CK56.3, and CK64.5) were randomly selected for further investigation. After being inoculated for three days, the leaves of C. kwangsiensi exhibited similar symptoms to those initially observed in the field, while no symptoms were observed on the control plants (Fig. 1). The fungi inoculated were consistently re-isolated from the necrotic tissue of the inoculated plants and identified based on their morphological characteristics and DNA sequence analysis (Additional file 1). No fungi were isolated from the plants in the negative control group.

Fig. 1.

(a–d) Symptoms of leaf spot disease on Curcuma kwangsiensis under natural conditions. (e–h) Pathogenicity testing on C. kwangsiensi seedlings. (e, f) Control; (g) Symptoms of the disease caused by strain CK56.3 on C. kwangsiensi after 3 days of inoculation; (h) Symptoms of the disease caused by strain CK56.3 on C. kwangsiensi after 9 days of inoculation.

Phylogenetic analyses

In this study, nine (9) new sequences of ITS, GAPDH and EF-1α were generated. The estimated sizes of individual alignments for ITS, GAPDH, and EF-1α were approximately 590 bp, 600 bp, and 990 bp correspondingly. To determine the phylogenetic position of our new isolates within the Curvularia genus, we conducted an analysis using a concatenated sequence dataset consisting of ITS, GAPDH, and EF-1α. In the phylogenetic analysis, the final concatenated alignment was 2180 bp. The topologies obtained from ML and BI analyses of the concatenated dataset were congruent (Fig. 2). The multilocus phylogenetic analysis showed the isolates CK43.1, CK56.3, CK64.5 are closely associated with C. bannonii but differ from it and other closely related taxa (Table 1). In the gene region of the ITS, the base sequence of C. sacchari-officinarum and C. guangxiensis was the same as our isolates, while C. bannonii, C. elliptiformis, C. clavata, and C. eleusinicola had 2 differences. Only C. eragrostidis had 3 base differences compared to our strains. In the gene region of EF-1α, the base sequence of C. bannonii matched our strains exactly, while there were 2 variations in C. elliptiformis and C. clavata, 3 variations in C. sacchari-officinarum, and 4 variations in C. eleusinicola and C. guangxiensis. GAPDH exhibited a range of 1 to 8 variations at the base level. In Curvularia, it appears that the mutation rate of DNA bases in the GAPDH gene region was evidently higher compared to the ITS and EF-1α gene regions.

Fig. 2.

The combined dataset of ITS, GAPDH, and EF-1α sequences was used to infer the phylogenetic tree of the genus Curvularia using Maximum Likelihood (ML) and Bayesian inference (BI). The tree was rooted with Bipolaris distoseptata (CGMCC 3.19361). The Ultrafast bootstrap values (left) greater than 70% and Bayesian posterior probabilities (right) exceeding 0.7 are indicated at the nodes. The examined isolates in present study are identified as the new species C. qinzhouensis, highlighted in red.

Table 1.

Pairwise comparison of the examined loci of newly identified species with closely related taxa.

Novel species	Closely related taxa	Loci, identity and percentage and gaps
		ITS		GAPDH		EF-1α
		Identity and percentage	Gaps	Identity and percentage	Gaps	Identity and percentage	Gaps
Curvularia qinzhouensis sp. nov. (CGMCC3.25225)	C. bannonii	395/397 (99.50%)	1/397 (0%)	581/586 (99.15%)	0/586 (0%)	804/804 (100%)	0/804 (0%)
	C. elliptiformis	539/541 (99.63%)	1/541 (0%)	591/599 (98.66%)	0/599 (0%)	924/926 (99.78%)	0/926 (0%)
	C. eragrostidis	571/574 (99.48%)	2/574 (0%)	553/554 (99.82%)	0/554 (0%)	–	–
	C. sacchari-officinarum	537/537 (100%)	0/537 (0%)	592/599 (98.83%)	0/599 (0%)	923/926 (99.68%)	0/926 (0%)
	C. clavata	378/380 (99.47%)	1/380 (0%)	491/496 (98.99%)	0/496 (0%)	802/804 (99.75%)	0/804 (0%)
	C. eleusinicola	565/567 (99.65%)	1/567 (0%)	577/585 (98.63%)	0/585 (0%)	895/899 (99.56%)	0/899 (0%)
	C. guangxiensis	531/531 (100%)	0/531 (0%)	594/599 (99.17%)	0/599 (0%)	922/926 (99.57%)	0/926 (0%)
	C. deserticola	588/592 (99.32%)	3/592(0%)	572/580 (98.62%)	0/580(0%)	913/916 (99.67%)	0/916(0%)

Taxonomy

Curvularia qinzhouensis H. Zhou & H.Y. Wang, sp. nov. (Fig. 3).

Fig. 3.

Morphological characteristics of the strain C. qinzhouensis CGMCC 3.25225: (a, b) Colony of the strain C. qinzhouensis on PDA; (c, d) Colony of the strain C. qinzhouensis on OA; (e, f) Colony of the strain C. qinzhouensis on MEA. (g, c–i) Conidia with conidiophore. (j) Conidia. Scale bars: g-i = 20 μm; j = 10 μm.

MycoBank accession number: MB# 849959.

Etymology. Name refers to the city in Guangxi Province where the strain was isolated.

Type. CHINA, Guangxi Province, Qinzhou City, on Curcuma kwangsiensis, May. 2021.

Hyphae branched, septate, smooth, 1.5–4 μm diam. Conidiophores arising singly or in groups, branched, straight or flexuous, septate, asperulate or smooth, with a thicker cell compared to vegetative hyphae, pale brown to dark brown in color, measuring 35.1-113.3 × 3.8–5.6 μm (average ± SD: = 72.6 ± 22.1 × 4.6 ± 0.5 μm, n = 30). Conidiogenous cells intercalary or terminal, sympodially proliferating, subcylindrical, smooth-walled, light brown to brown, measuring 4.8–32.1 × 3.2–6.7 μm (average ± SD: 17.6 ± 7.7 × 4.7 ± 0.7 μm, n = 50) Conidia usually ellipsoidal, 2–3 septate, central cells slightly larger, apical and basal cells slightly paler compared to the central cells, middle septum truly median, often thick and dark, smooth, dark brown, size 15.5–24.1 × 7.4–12.8 μm (average ± SD: 19.3 ± 1.9 × 9.9 ± 1.1 μm, n = 60). Chlamydospores not observed. Hilum slightly protruding thickened, darkened.

Colonies on PDA grown in the dark exhibited rapid growing, attaining 8.2 cm diam after 7 days at 28 °C, colony flat, entire edge, aerial mycelia dense, greenish black; reverse blackish green. Colonies on OA grown in the dark reaching 8.4 cm diam after 7 days at 28 °C, flat, dense, circular with entire edge, gray-green; reverse olive-green. Colonies on MEA grown in the dark reaching 7.9 cm diam in after days at 28 °C, flat, dense, circular with entire edge, gray-brown; reverse blackish green.

Notes: Curvularia qinzhouensis is most closely associated with C. bannonii [MB#135463] and other members within the sibling clade, i.e. C. elliptiformis [MB#556658], C. eragrostidis [MB#296246], C. sacchari-officinarum [MB#556670], C. eleusinicola [MB#835142], C. deserticola [MB#853988] and C. clavata [MB#329439]^14–17. While, C. qinzhouensis differs from C. bannonii and C. eragrostidis in having shorter conidiophore (35.1–113.3 μm vs. about 950 μm and 90–710 μm, respectively)^15,18. C. qinzhouensis can be distinguished by the shape or size of its conidia (15.5–24.1 × 7.4–12.8 μm for ellipsoidal shape) compared to those in C. bannonii (24-34.9 × 13–17 μm) and C. clavata (14.5–29 × 4–9 μm for clavate shape)¹⁵. C. qinzhouensis differs from C. guangxiensis in conidia size and colony appearance. C. qinzhouensis has ellipsoidal conidia measuring 15.5–24.1 × 7.4–12.8 μm, while C. guangxiensis has ovoid or globose/subglobose conidia ranging from 12 to 24 μm in length and 9–18 μm in width. Moreover, C. qinzhouensis forms dense, greenish-black colonies on PDA and flat, gray-green colonies on OA, while C. guangxiensis has sparse, light grey colonies on PDA and raised, light grey colonies on OA. C. qinzhouensis differs from C. elliptiformis and C. sacchari-officinarum in its conidial and colony characteristics. Specifically, C. qinzhouensis has smooth conidia with paler apical and basal cells, a thick, dark middle septum, and a slightly protruding hilum. C. elliptiformis has conidia with one pale brown cell at the apex or base, three noticeably brown cells, a lighter, thinner middle septum, and a non-protruding hilum. C. sacchari-officinarum has slightly verruculose conidia with a lighter, thinner middle septum and a non-protruding hilum¹⁵. On PDA, C. qinzhouensis forms flat, greenish black colonies, C. elliptiformis has slightly radiating margins and color variation, and C. sacchari-officinarum shows slightly raised colonies with varying colors. On OA, C. qinzhouensis forms flat, gray-green colonies, C. elliptiformis forms crateriform white colonies, and C. sacchari-officinarum forms gray-white colonies¹⁵. C. qinzhouensis differs from C. eleusinicola in its shorter conidiophore (35.1–113.3 μm vs. ~150 μm). Additionally, C. qinzhouensis has entire edges and greenish-black colonies, while C. eleusinicola has undulate margins, moderate aerial mycelia, a dark brown center, pale brown periphery, and dark brown concentric rings¹⁸. C. qinzhouensis differs from C. deserticola by its longer conidiophore cells (4.8–32.1 μm vs. 4–6 μm). Moreover, C. qinzhouensis forms dense, greenish-black colonies on PDA and gray-green colonies on OA, while C. deserticola has olive to silver colonies with sparse aerial mycelia on PDA and brown colonies with olive-colored centers on OA¹⁷. C. clavata has been documented on various plant species in different regions, including Eleusine coracana in Sri Lanka, Dactyloctenium aegyptium in Australia, Oryza sativa in India, Saccharum officinarum in Guyana, Curcuma wenyujin in China, and Tobacco in China^18–20.C. elliptiformis and C. sacchari-officinarum have been reported on Saccharum officinarum in China¹⁵. C. bannonii has been found on Stipagrostis ciliate and Jacquemontia tamnifolia in the USA^14,17. C. eragrostidis has been observed on Sorghum seed in Java²¹.

Biological characterization of C. Qinzhouensis

Previous studies have demonstrated the significant impact of various factors, such as temperature and acidity, on the proliferation and virulence of pathogens^22,23. In the case of C. qinzhouensis, Fig. 4a shows that it can grow within a temperature range of 10–40 °C. Among these temperatures, the highest growth rate was observed at 30 °C, resulting in colony diameters of approximately 89.0 ± 1.00 mm after 6 days of incubation. Slower colony growth was observed at 15 °C, 20 °C, 38 °C, and 40 °C. The slowest growth occurred at 10 °C, with colony diameters of approximately 15.6 ± 0.60 mm after 6 days of incubation. These findings indicate that C. qinzhouensis thrives best within the temperature range of 25–35 °C, which is considered the most favorable temperature range.

Fig. 4.

Mycelial growth of C. qinzhouensis under different pH and temperature on PDA. (a) Temperature. (b) pH. The data are presented as the mean ± SD (n = 3 biologically replicates). The different letters above the columns signify a significant difference (p < 0.05, one-way ANOVA with Duncan’s multiple range test) in the data among the groups.

This study reveals that C. qinzhouensis is capable of growing within a pH range of 4 to 11, as depicted in Fig. 4b. At pH 11, the mycelia exhibited slow growth, resulting in small and scattered colony diameters measuring 56.5 ± 0.84 mm. Conversely, the colony displayed the highest growth rate at pH 8, with diameters of 79.2 ± 1.17 mm. There were no significant differences in growth between the experimental groups at pH values of 6 and 7. These findings suggest that C. qinzhouensis thrives in both acidic and mildly alkaline environments but experiences hindered growth under highly alkaline conditions.

Antimicrobial activity of phytochemicals against mycelial growth

This study highlights the potential benefits of examining phytochemicals for the development of environmentally friendly and non-toxic fungicides to effectively manage C. qinzhouensis. Results from sensitivity testing, presented in Table 2; Fig. 5, indicate a strong correlation (correlation coefficients > 0.90) between phytochemical dosage and inhibition of C. qinzhouensis. Notably, Honokiol exhibited significant antifungal activity with half-maximal effective concentration (EC₅₀) value of 6.72 ± 1.75 µg/ml. Thymol and citral demonstrated EC₅₀ values of 25.74 ± 4.30 µg/ml and 54.24 ± 4.69 µg/ml, respectively. These were followed by geraniol, with EC₅₀ values of 77.09 ± 10.83 mg/l. Conversely, carvone and citronellal displayed the lowest antifungal activity against the pathogen, with EC₅₀ values of 161.53 ± 22.47 mg/ml and 134.00 ± 30.37 mg/ml, respectively.

Table 2.

The activity of phytochemicals in inhibiting the growth of C. Qinzhouensis.

Phytochemicals	Concentration (µg/ml)	Regression equation	EC50 (µg/mL)	Coefficient of determination (R2)
Geraniol	10, 20, 50, 100, 150	y = 2.077x + 0.4268	77.09 ± 10.83	0.9805
Carvone	50, 100, 150, 200, 250	y = 2.322x + 0.2854	161.53 ± 22.47	0.9649
Citral	25, 50, 100, 200, 500	y = 1.550x + 1.883	54.24 ± 4.69	0.9302
Thymol	25, 30, 40, 50, 100	y = 2.039x + 2.201	25.74 ± 4.30	0.9795
Citronellal	30, 60, 90, 120, 150	y = 2.031x + 0.2508	134.00 ± 30.37	0.9698
Honokiol	2, 5, 10, 20, 30	y = 1.557x + 3.530	6.72 ± 1.75	0.9801

Fig. 5.

Effects of different phytochemicals on the growth of C. qinzhouensis. (a) Mycelial growth inhibition of C. qinzhouensis CK56.3 after the application of different phytochemicals under a series of concentrations. (b) Effects of six phytochemicals on the mycelial growth of C. qinzhouensis after 3 days in vitro. The data are presented as the mean ± SD (n = 3 biologically replicates). The different letters above the columns signify a significant difference (p < 0.05, one-way ANOVA with Duncan’s multiple range test) in the data among the groups.

Discussion

The three isolates in this study exhibited morphological similarities with five known Curvularia species: C. bannonii, C. elliptiformis, C. eragrostidis, C. radici-foliigena, and C. clavata^14–16. However, they also displayed slight differences that did not correspond to any of these species. The key morphological characteristics that distinguish our strains from related species were the dimensions of conidiophores and conidia. The conidial length of C. qinzhouensis was very close to C. eragrostidis (15.5–24.1 × 7.4–12.8 μm vs. 13.8–24.0 × 7.6–13.6 μm), while C bannonii produced the larger conidia (24-34.9 × 13–17 μm). Conidiophores of C. qinzhouensis were shorter than those of C. bannonii and C. eragrostidis¹⁴. Nevertheless, accurate microscopic identification of these organisms remains challenging due to overlapping physical characteristics. To address this, we employed a multi-locus analysis utilizing ITS, GAPDH, and EF-1α gene sequences, which also confirmed that these isolates belong to a novel species with strong bootstrap support (Fig. 2). Furthermore, we conducted a comparison of the DNA sequence similarity of the ITS, GAPDH, and EF-1α gene regions (Table 1). The EF-1α gene of C. bannonii had the same base sequence as our strains, except for a single bp difference in the ITS gene region. However, in the GAPDH gene regions, there were 5 bp difference characters observed between C. qinzhouensis and C. bannonii. Notably, within the Curvularia genus, GAPDH exhibits a higher rate of evolution compared to ITS and EF-1α.

Curvularia is a widely distributed genus known for causing various plant diseases⁴. Curvularia infection are common fungal diseases. For instance, C. lunata infection causes leaf spots on maize²⁴ and root rot on Strawberry²⁵. Leaf spot on Sansevieria trifasciata¹¹ and postharvest rot on Pineapple²⁶ can be caused by C. eragrostidis, while C. hawaiiensis infection leads to leaf spot on rice²⁷. In this study, the pathogenicity analysis revealed that diseased seedlings exhibited yellow, watery, and irregular lesions on their leaves. As the infection progressed, the entire leaf dried up, turned yellow, and eventually perished. To our knowledge, this is the first report of leaf spot on C. kwangsiensi caused by C. qinzhouensis worldwide. Investigating the optimal growth temperature and pH for pathogenic fungi helps understand their ecological characteristics, transmission pathways, and aids in the development of effective control measures. The pathogen thrives and grows rapidly in warmer environments (25–35 °C), suggesting a higher likelihood of causing plant diseases during warmer seasons. The fungus shows adaptability to higher pH levels (pH 8), indicating its ability to survive and reproduce better in alkaline soil or alkaline environments. As a newly discovered species of Curvularia, further investigation is needed to determine the host range of C. qinzhouensis.

Phytochemicals derived from natural sources have gained attention for their potential in managing plant diseases. These compounds offer advantages such as minimal toxicity, low potential for fungicide resistance, and suitability for organic agriculture²⁸. In our study, we tested six phytochemical agents for their antifungal properties against C. qinzhouensis. Honokiol, thymol, and citral were found to effectively suppress the growth of the fungus. On the other hand, carvone and citronellal showed the least effective control. Further research is needed to fully understand the antifungal effects of these phytochemical agents, and their efficacy in controlling leaf diseases should be evaluated under field conditions.

Materials and methods

Sample collection and fungal isolation

We obtained the permission to collect symptomatic plants of C. kwangsiensisi from private plantations, Qinzhou City, Guangxi Province, China in 2021. The plants were identified by Dr. Qi Gao (Professor, Plant resources Conservation and Utilization Laboratory, Guangxi Minzu University, Nanning, China), assigned voucher no. CK # 2021050022 and were deposited in the Virtual Herbarium, Plant resources Conservation and Utilization Laboratory, Guangxi Minzu University, Nanning, China. The latitude of the sampling place is 21.51°N and longitude is 108.44°E. To investigate the disease, six randomly selected symptomatic plants were subsequently transported to the laboratory for analysis. Tissue isolation and single spore purification methods were employed to obtain isolates associated with leaf spot. In brief, plant samples were cleaned and cut into small pieces (~ 5 mm), followed by surface sterilization using 75% ethanol for 2 min. Subsequently, they were soaked in a 1% sodium hypochlorite solution for 60 s and rinsed three times with sterile distilled water for 60 s each. The tissues were placed on potato dextrose agar (PDA, containing 200 g/L potato, 20 g/L glucose and 20 g/L agar) and incubated in the dark at 28 °C for 3 days. Cultures were obtained by transferring hyphal tips from edge of colonies to fresh PDA. For single spore purification, the fungal material was collected using a sterilized inoculating needle and placed in sterilized distilled water. The material was then mechanically disrupted to obtain a suspension of spores. After being spread on water agar (WA, containing 20 g/L agar), the spore suspension was incubated overnight at 28 °C. Germinated spores were marked under a microscope and then moved to PDA and cultivated at 28 °C to obtain pure cultures^29,30. The pure cultures were stored at 4 °C. in the Microbiology laboratory, Guangxi Minzu University, China.

Pathogenicity assay

To confirm the pathogenicity of the fungi, nine healthy seedlings of C. kwangsiensi that had been growing for four weeks, were inoculated with mycelial plugs (5 mm in diameter) cut from the edges of actively growing cultures cultivated on WA in the dark at 28 °C for 5 days. As a control, three plants were treated with sterile WA. The greenhouse maintained a temperature of 35 °C with a 12-hour photoperiod and around 90% relative humidity for all the inoculated plants. Following a three-day period of incubation, the inoculated plants were monitored for the development of symptoms. The experiments were repeated three times. Infected leaves were collected and the pathogens were re-isolated using the tissue isolation method. Morphological and sequencing analyses were conducted to compare the re-isolated pathogens with the original strains.

DNA extraction, sequencing and phylogenetic analyses

Genomic DNA was extracted from freshly mycelia grown on PDA using the CTAB method³¹, with a slight modification. The extracted DNA was then stored at -20 °C. PCR amplification of ITS region was performed using the primer pair ITS1 and ITS4. For GAPDH amplification, the primer pair GPD-1 and GPD-2 was utilized, while EF-1α was amplified with the primer pair EF1-983 F and EF1-2218R, as specified in Table 3.

Table 3.

Primers and references used in this study.

Gene	Primer	Sequence (5′ → 3′)	References
ITS	ITS1	TCC GTA GGT GAA CCT GCG G	32
	ITS4	TCC TCC GCT TAT TGA TAT GC
GAPDH	GPD-1	CAA CGG CTT CGG TCG CAT TG	33
	GPD-2	GCC AAG CAG TTG GTT GTG C
EF-1α	EF1-983 F	GCY CCY GGH CAY CGT GAY TTY AT	34
	EF1-2128R	AT GAC ACC RAC RGC RAC RGT YTG

The PCR reaction mixture was prepared with a total volume of 25 µL. It contained 2.5 µL of 10 × PCR reaction Buffer, 125 µM of each dNTP, 0.4 µM of each primer, 0.1 µL of EasyTaq DNA polymerase (Trans gene, Beijing, China), and 1 ng of genomic DNA. The PCR parameters for amplifying ITS, GAPDH, and EF-1α were as follows: an initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 90 s, with a final elongation step of 10 min at 72 °C. The final PCR products were analyzed by electrophoresis in 1% agarose gel. The PCR samples were sent to a commercial provider (Audio Codes Dingsheng Biotechnology Co., Ltd, Wuhan) for Sanger sequencing.

In this study, the phylogenetic analysis comprised the isolates CK43.1, CK56.3, CK64.5, as well as reference isolates representing a total of 64 Curvularia species (Table 4). Strain CGMCC 3.19361 of Bipolaris distoseptata was used as the outgroup. Phylogenetic tree construction was performed using PhyloSuite software³⁵ through Bayesian inference (BI) and Maximum Likelihood (ML) methods, based on a combined dataset of ITS, GAPDH, and EF-1α sequences. Nodes are indicated with Ultrafast bootstrap values (left) that are higher than 70% and Bayesian posterior probabilities (right) that surpass 0.7.

Table 4.

Strains and their GenBank accession numbers used in the present study.

Species	Strain accession	GenBank accession
		ITS	GAPDH	EF-1α
Curvularia akaiiensis	BRIP 16,080 T	KJ415539	KJ415407	KJ415453
Curvularia alcornii	MFLUCC 10–0703 T	JX256420	JX276433	JX266589
Curvularia asianensis	MFLUCC 10–0711 T	JX256424	JX276436	JX266593
Curvularia australiensis	BRIP 12,044 T	KJ415540	KJ415406	KJ415452
Curvularia australis	BRIP 12,521 T	KJ415541	KJ415405	KJ415451
Curvularia bannonii	BRIP 16,732 IsoT	KJ415542	KJ415404	KJ415450
Curvularia beasleyi	BRIP 10,972 T	MH414892	MH433638	MH433654
Curvularia beerburrumensis	BRIP 12,942 T	MH414895	MH433634	MH433657
Curvularia boeremae	IMI 164,633 T	MH414911	MH433641	–
Curvularia borreriae	CBS 859.73	HE861848	HF565455	–
Curvularia bothriochloae	BRIP 12,522 T	KJ415543	KJ415403	KJ415449
Curvularia clavata	BRIP 61680b	KU552205	KU552167	KU552159
Curvularia coatesiae	BRIP 24,261 T	MH414897	MH433636	MH433659
Curvularia coicis	CBS 192.29 SynT	AF081447	AF081410	JN601006
Curvularia colbranii	BRIP 13,066 T	MH414898	MH433642	MH433660
Curvularia cymbopogonis	CBS 419.78	HG778985	HG779129	–
Curvularia crustacea	BRIP 13,524 ET	KJ415544	KJ415402	KJ415448
Curvularia dactyloctenii	BRIP 12,846 T	KJ415545	KJ415401	KJ415447
Curvularia deserticola	CMW 58,190 T	ON074985	ON355399	ON355360
Curvularia deserticola	CMW 64,024	ON075008	ON355400	ON355361
Curvularia dactyloctenicola	CPC 28,810 T	MF490815	MF490837	MF490858
Curvularia eleusinicola	USJCC-0005 T	MT262877	MT393583	MT432925
Curvularia elliptiformis	CGMCC 3.19351 T	MN215656	MN264091	MN263950
Curvularia elliptiformis	LC12004	MN215659	MN264092	MN263953
Curvularia ellisii	CBS 193.62 T	JN192375	JN600963	JN601007
Curvularia eragrostidis	CBS 189.48	HG778986	HG779154
Curvularia eragrosticola	BRIP 12,538 T	MH414899	MH433643	MH433661
Curvularia falsilunata	CGMCC 3.19329 T	MN215660	MN264093	MN263954
Curvularia gladiolii	ICMP 6160	JX256426	JX276438	JX266595
Curvularia graminicola	BRIP 23,186 T	JN192376	JN600964	JN601008
Curvularia guangxiensis	CGMCC 3.19330 T	MN215667	MN264100	MN263961
Curvularia guangxiensis	LC11997	MN215668	MN264104	MN263965
Curvularia guangxiensis	LC12000	MN215671	MN264138	MN264000
Curvularia harveyi	BRIP 57,412 T	KJ415546	KJ415400	KJ415446
Curvularia hawaiiensis	BRIP 11,987 IsoLT	KJ415547	KJ415399	KJ415445
Curvularia heteropogonicola	BRIP 14,579 IsoLT	KJ415548	KJ415398	KJ415444
Curvularia heteropogonis	CBS 284.91 T	KJ415549	JN600969	JN601013
Curvularia hominis	CBS 136,985 T	HG779011	HG779106	–
Curvularia homomorpha	CBS 156.60 T	JN192380	JN600970	JN601014
Curvularia intermedia	CBS 334.64	HG778991	HG779155	–
Curvularia ischaemi	CBS 630.82 T	HG778992	HG779131	–
Curvularia kenpeggii	BRIP 14,530 T	MH414900	MH433644	MH433662
Curvularia kusanoi	CBS 137.29	JN192381	–	JN601016
Curvularia lamingtonensis	BRIP 12,259 T	MH414901	MH433645	MH433663
Curvularia lonarensis	CBS 140,569 T	KT315408	KY007019	–
Curvularia lunata	CBS 730.96 NeoT	JX256429	JX276441	JX266596
Curvularia mebaldsii	BRIP 12,900 T	MH414902	MH433647	MH433664
Curvularia microspora	GUCC 6272 T	MF139088	MF139106	MF139115
Curvularia mosaddeghii	IRAN 3131 C T	MG846737	MH392155	MH392152
Curvularia muehlenbeckiae	CBS 144.63 T	HG779002	HG779108	–
Curvularia neergaardii	BRIP 12,919 IsoT	KJ415550	KJ415397	KJ415443
Curvularia nicotiae	CBS 655.74 T	KJ909772	KM083614	–
Curvularia sacchari-officinarum	CGMCC 3.19331 T	MN215705	MN264137	MN263998
Curvularia sacchari-officinarum	LC12008	MN215707	MN264138	MN264000
Curvularia ovariicola	CBS 470.90 T	JN192384	JN600976	JN601020
Curvularia papendorfii	BRIP 57,608 T	KJ415552	KJ415395	KJ415441
Curvularia perotidis	BRIP 13,466 T	JN192385	KJ415394	JN601021
Curvularia portulacae	BRIP 14,541 T	KJ415553	KJ415393	KJ415440
Curvularia portulacae	BRIP 14,837 T	KJ415554	KJ415392	KJ415439
Curvularia qinzhouensis sp. nov.	CGMCC 3.25225 = CK56.3	OR575729	OR576898	OR576895
Curvularia qinzhouensis sp. nov.	CK43.1	OR575728	OR576897	OR576894
Curvularia qinzhouensis sp. nov.	CK64.5	OR575730	OR576899	OR576896
Curvularia radici-foliigena	CGMCC3.19328 T	MN215695	MN264127	MN263988
Curvularia ravenelii	BRIP 13,165 T	JN192386	JN600978	JN601024
Curvularia richardiae	BRIP 4371 T	KJ415555	KJ415391	KJ415438
Curvularia ryleyi	BRIP 12,554 T	KJ415556	KJ415390	KJ415437
Curvularia sorghina	BRIP 15,900 T	KJ415558	KJ415388	KJ415435
Curvularia spicifera	CBS 274.52 T	JN192387	JN600979	JN601023
Curvularia tripogonis	BRIP 12,375 IsoT	JN192388	JN600980	JN601025
Curvularia tropicalis	BRIP 14,834 T	KJ415559	KJ415387	KJ415434
Curvularia tuberculata	CBS 146.63 IsoT	JX256433	JX276445	JX266599
Bipolaris distoseptata	CGMCC 3.19361 T	MN215628	MN264064	MN263922

‘–’ indicates the absence of GAPDH or EF-1α genes in the GenBank accession, where T represents the Ex-type.

Morphological characterization

The examined isolates were cultured on PDA at 28 °C for 7 days in darkness. Round mycelial discs with a diameter of 5 mm were then obtained from the colony periphery and transferred to various fresh media for morphological examinations. The cultural characteristics of the isolates were analyzed by incubating them for 7 days at 28 °C in darkness on PDA, malt extract agar (MEA, containing 30 g/L malt extract and 20 g/L agar), and oatmeal agar (OA, containing 30 g/L oatmeal and 20 g/L agar). Growth rate determination was performed by measuring the radial colonial diameters in at least four different directions after 7 days of incubation at 28 °C in darkness³⁶. Conidia were induced on WA medium, and the features of both conidia and conidiophores were observed and documented using a digital microscope (DM2000, Leica, Germany).

Biological characteristics of the pathogen

The isolates were cultured on PDA with pH values ranging from 4 to 11 at 28 °C. The pathogen-inoculated PDA (pH 7.0) plates were incubated at different temperatures in an artificial climate chamber, including 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 28 °C, 30 °C, 35 °C, and 38 °C. The growth of the colonies was monitored over a period of 6 days, with colony diameters measured using the crossover method and documented through photography³⁷. Each experiment was repeated three times with two replicates in each.

Antimicrobial activity of phytochemicals against mycelial growth

To identify phytochemicals with effective control against the pathogen, various phytochemicals were screened. Six plant compounds provided by Macklin Biochemical Technology Co., Ltd. (China), including geraniol, citral, thymol, citronellal (dissolved in ethanol), honokiol (dissolved in dimethyl sulfoxide), and carvone/magnolol (dissolved in acetone), were used as the test substances³⁸. Initially, the phytochemicals were dissolved in 1 ml of the suitable solvent, subsequently mixed with water, and introduced into the PDA medium (cooled to approximately 50 °C) to prepare a range of concentration gradients. Each treatment had three replicates. Mycelial disks (5 mm in diameter) were transferred from the colony edge to the center of the PDA plates, which were then incubated in the dark at 28 °C. After 3 days, the colony diameter was measured using a ruler. The experiments were repeated three times with two replicates in each.

The inhibition rate I (%) was then calculated using the provided formula, where C (cm) and T (cm) indicate the tested fungi diameters of the control and treated PDA plates, respectively. The regression equation, determination coefficient (R²), EC₅₀ values of inhibiting mycelial growth for various phytochemicals were determined using the software GraphPad Prism 8.0.

Statistical analysis

Data analyses were conducted using Excel 2016 and GraphPad Prism 8. GraphPad Prism 8 was employed to perform normality and homogeneity of variance tests. To determine the significance of differences, a one-way ANOVA with Duncan’s multiple range test was conducted in GraphPad Prism 8, with a significance level of p < 0.05. Graphs were generated using GraphPad Prism 8.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization and methodology, H.W. and R.W.; validation and formal analysis, J.W. and J.S.; investigation, H.W., J.X., M.X., and D.T.; writing—original draft preparation, H.W.; writing—review and editing, H.W. and H.Z.; project administration and funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China, grant number 32060599; the National Natural Science Foundation of China, grant number 81860678; and the Natural Science Foundation of Guangxi Province, grant number 2021GXNSFAA075023.

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.