Curvularia lingshanensis sp. nov., a New Pathogen Causing stem and leaf rot on Curcuma kwangsiensis and Its Sensitivity to Five Fungicides

doi:10.21203/rs.3.rs-3594908/v1

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Curvularia lingshanensis sp. nov., a New Pathogen Causing stem and leaf rot on Curcuma kwangsiensis and Its Sensitivity to Five Fungicides

https://doi.org/10.21203/rs.3.rs-3594908/v1

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Curcuma kwangsiensis S. G. Lee et C. F. Liang (Zingiberaceae) is a traditional Chinese medicinal plant that is cultivated in the South-West of China. In this study, five fungal strains were isolated from diseased C. kwangsiensis plants in Lingshan County, Guangxi Province. The phylogenetic analyses, based on a combined dataset of ITS, GAPDH, and EF-1α gene sequences, revealed that the strains from this study formed a distinct clade separate from other known Curvularia species. Additionally, these strains were closely related to C. tamilnaduensis, C. radici-foliigena, and C. radicicola. Based on morphological observations, the strains exhibit distinct variations in conidiophore length, conidiogenous cell characteristics, as well as conidial size and shape, distinguishing them from similar species. Consequently, these five strains have been classified as a novel species, C. lingshanensis. Pathogenicity test carried out on the plants confirmed that the new species was pathogenic to C. kwangsiensis. Five fungicides were tested for their toxicity to the pathogen using the mycelial growth rate method. Results indicated that among the studied fungicides, 70% mancozeb, 10% difeno-conazole and 75% trifloxystrobin·tebuconazole had the highest antifungal activity against C. lingshanensis with EC50 values of 1.5233 ± 0.18 mg/L, 0.1748 ± 0.03 mg/L, 0.2557 ± 0.06 mg/L, respectively. This study provides the first report of a severe stem and leaf rot disease on C. kwangsiensis caused by C. lingshanensis and important guidance for preventing and controlling this disease effectively.

Curcuma kwangsiensis

stem and leaf rot

new Curvularia species

molecular identification

fungicide.

Curcuma kwangsiensis S. G. Lee et C. F. Liang, belongs to the Zingiberaceae family, is an important perennial medicinal plant widely cultivated in south-western China(Xin et al. 2022). The tubers and rhizomes of C. kwangsiensis have a wide range of medical value, and are commonly used for treating cancer, cardiovascular disease, asthma and inflammation in traditional Chinese medicine(Zhou et al. 2018; Kaijing et al. 2022). According to recent studies, it has a wide range of pharmacological activities, including antitumor, antioxidation, blood lipid reduction and immunosuppression(ZeYu et al. 2022). With the deepening of research on medicinal functions, the demand for C. kwangsiensis is growing. Therefore, the cultivation areas of C. kwangsiensis have increased significantly, and the disease severity caused by fungi has also increased when plants were cultivated in suboptimal environment. For example, Epicoccum latusicollum can cause the leaf blight on C. kwangsiensis, which leads to defoliation eventually(Hanyi et al. 2023). However, at present, the researches on C. kwangsiensis mainly focus on chemistry and pharmacology(Ju-Tao et al. 2019; Lian et al. 2020). Most of the pathogens that cause diseases in C. kwangsiensis are unknown.

The Curvularia genus includes many endophytic fungi and pathogens of plants, animals and humans(F. et al. 2022; Jason et al. 2022). Curvularia species, as the plant pathogens, are distributed worldwide and infect a wide range of crops (Marin-Felix et al. 2017). Curvularia was traditionally identified at genus level by examining their pigmentation, septate conidia, and hyphae morphology. However, it is difficult to accurately identify the members of the Curvularia genus solely through morphology, such as size, shape, and septum of conidia, without DNA molecular characterization(Madrid et al.). Multi-gene sequence analysis based on the internal transcribed spacer (ITS) region of rDNA, the partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the partial translation elongation factor (EF-1α) regions is usually used to classify species within the Curvularia genus (Manamgoda et al. 2012; Iturrieta-González et al. 2020; Wei et al. 2022).

In 2021, a serious stem and leaf rot disease was observed on Curcuma kwangsiensis in Guangxi province. In order to determine the pathogen causing the stem and leaf rot of C. kwangsiensis, this study used both tissue and single spore isolation methods to isolate and purify the pathogen from the symptomatic plants, and then determined their pathogenicity according to Koch’s Rule. Based on morphological descriptions and phylogenetic analyses, the isolates have been identified as a new Curvularia species. Our study provides further insight into the Curvularia disease in C. kwangsiensis in China. This study provides further insight into Curvularia disease in C. kwangsiensis in China and provides important guidelines for preventing and controlling this disease.

Isolation

In May 2021, a stem and leaf rot disease was observed on Curcuma kwangsiensi grown in a plantation (21°51'00″N, 108°44'00″E) in Guangxi Province. In total, three plants with typical stem and leaf rot disease were collected from different locations and transported to the laboratory for further examination. The plant samples were washed and cut into small pieces (approximately 5 × 5 mm), surface sterilized in 75% ethanol for two minutes, soaked for 60 seconds in 1% sodium hypochlorite (NaOCl) solution, and then rinsed for 60 seconds in sterile water three times. The pieces were plated onto potato dextrose agar (PDA) and cultured at 28°C for 3 days. Mycelium was transferred from colonies edges to a fresh PDA plate for further culturing. For single spore isolation, sterilized inoculating needles were used to collect fungal content, which was then soaked in sterilized water, and then mechanically broken up to obtain suspensions of spores. A suspension of spores was spread onto water agar (WA) and incubated overnight at 28°C. In order to obtain pure cultures, we marked germinated spores under a microscope, transferred them to PDA media aseptically, and cultured them for 48 hours at 28°C (Zhang et al. 2013). The obtained pure cultures were stored at 4°C.

Morphological studies

For morphological observation, mycelial discs (5 mm diameter) were cut from the edges of colonies and transferred to media for incubation at 28°C in darkness for 7 days. The isolates culture characteristics were observed on PDA, oatmeal agar (OA) and malt extract agar (MEA), after 7 days of incubation at 28°C in the dark. For growth rate determination, plates were incubated at 28°C in the dark and growth rates were calculated after 7 days of incubation by measuring radial colonial growth in at least four different directions(M et al. 2019). Conidia were induced on water-agar medium (WA). Conidia and conidiophore characteristics were assessed using a digital microscope (DM2000, Leica, Germany). The characteristics of the colonies were recorded. As described previously, conidia and conidiophore characteristics were determined (Daliyamol et al. 2021).

DNA Extraction and Sequencing

Genomic DNA was was extracted from fungal mycelia growing on PDA by the cetyltrimethyl ammonium bromide (CTAB) method (Porebski et al. 1997), with a slight modification, and stored at − 20°C. The ITS, GAPDH, and EF-1α gene regions were amplified using primers ITS1/ITS4, GPD-1/GPD-2, and EF-983F/EF1-2128R, respectively (Table 1). There was a volume of 25 µL in the PCR reaction, including 2.5 µL 10 × PCR Buffer (Trans gene, Beijing, China), 0.5 µL dNTPs, 0.5 µL of each forward and reverse primer, 0.1 µL Taq DNA polymerase, 20.9 µL sterile water and 1 ng genomic DNA. The PCR procedures for ITS, GAPDH and EF-1α were done under the following conditions: predenaturation at 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 55°C for 60 s and 72°C for 90 s with a final extension of 10 min at 72°C. The final PCR products were determined by electrophoresis in 1% agarose gel. Sequencing of the PCR products was performed by a company (Audio Codes Dingsheng Biotechnology Co., Ltd, Wuhan).

Table 1

Primers used in this study.
Gene	Primer	Sequence (5′ → 3′)	References
ITS	ITS1	TCC GTA GGT GAA CCT GCG G	(White et al. 1990)
ITS	ITS4	TCC TCC GCT TAT TGA TAT GC	(White et al. 1990)
GAPDH	GPD-1	CAA CGG CTT CGG TCG CAT TG	(Berbee 1999)
GAPDH	GPD-2	GCC AAG CAG TTG GTT GTG C	(Berbee 1999)
EF-1α	EF-983F	GCY CCY GGH CAY CGT GAY TTY AT	(Ignazio et al. 1999)
EF-1α	EF1-2128R	AT GAC ACC RAC RGC RAC RGT YTG	(Ignazio et al. 1999)

The phylogenetic analysis included the five strains in this study and reference isolates representing 47 species of Curvularia. Strain BRIP 12790 of Bipolaris peregianensis was used as the outgroup (Table 2). A phylogenetic tree was constructed based on a concatenated dataset (ITS + GAPDH + EF-1α) with Bayesian inference (BI) and Maximum Likelihood (ML) methods in PhyloSuite software(Dong et al. 2020). At the nodes, ultrafast bootstrap values (left) above 70% and Bayesian posterior probabilities (right) above 0.8 are indicated.

Table 2

Strains used in the present study and their GenBank accession numbers.
Species	Strain Accession	GenBank Accession
Species	Strain Accession	ITS	GAPDH	EF-1α
Curvularia aeria	CBS 294.61 T	JX256420	JX276433	JX266589
C. alcornii	MFLUCC 10–0703 T	JX256420	JX276433	JX266589
C. australiensis	BRIP 12044 T	KJ415540	KJ415406	KJ415452
C. australis	BRIP 12521 T	KJ415541	KJ415405	KJ415451
C. asianensis	MFLUCC 10–0711 T	JX256424	JX276436	JX266593
C. americana	UTHSC 08-3414 T	HE861833	HF565488	–
C. carica-papayae	CBS 135941 T	HG778984	HG779146	–
C. chlamydospora	UTHSC 07-2764 T	HG779021	HG779151	–
C. coimbatorensis	SZMC 22225 T	MN628310	MN628306	MN628302
C. dactyloctenii	BRIP 12846 T	KJ415545	KJ415401	KJ415447
C. eragrostidis	ZGZ990780	HM053668	HM053657	–
C. gladiolii	ICMP 6160	JX256426	JX276438	JX266595
C. graminicola	BRIP 23186 T	JN192376	JN600964	JN601008
C. hawaiiensis	BRIP 11987 IsoT	KJ415547	KJ415400	KJ415445
C. heteropogonis	CBS 284.91 T	JN192379	KJ415398	JN601013
C. hominis	CBS 136985 T	HG779011	JN600969	–
C. homomorpha	CBS 156.60 T	JN192380	HG779106	JN601014
C. heteropogonicola	BRIP 14579 IsoT	KJ415548	KJ415398	KJ415444
C. intermedia	CBS 334.64	HG778991	KM061787	–
C. ischaemi	CBS 630.82 T	HG778992	HG779131	–
C. kusanoi	CBS 137.29	JN192381	MH433644	JN601016
C. lingshanensis sp. nov.	CGMCC3.25224 = CK21.1	OR575881	OR576905	OR576900
C. lingshanensis sp. nov.	CK21.6	OR575882	OR576906	OR576901
C. lingshanensis sp. nov.	CK21.9	OR575883	OR576907	OR576902
C. lingshanensis sp. nov.	CK21.14	OR575884	OR576908	OR576903
C. lingshanensis sp. nov.	CK21.18	OR575885	OR576909	OR576904
C. lonarensis	CBS 140569 T	KT315408	MH433645	–
C. lunata	CBS 730.96 NeoT	JX256429	KY007019	JX266596
C. neergaardii	BRIP 12919 IsoT	KJ415550	KJ415397	KJ415443
C. nodulosa	CBS 160.58	JN601033	JN600975	JN601019
C. nicotiae	BRIP 11983	KJ415551	KJ415396	KJ415442
C. ovariicola	CBS 470.90 T	JN192384	JN600976	JN601020
C. papendorfii	CBS 308.67 T	KJ415552	KJ415395	KJ415441
C. perotidis	CBS 350.90 T	JN192385	KJ415394	JN601021
C. portulacae	CBS 239.48 IsoT	KJ909775	KM083616	–
C. petersonii	BRIP 14642 T	MH414905	MH433650	MH433668
C. protuberans	CGMCC 3.19360 T	MN215693	MN264125	MN263986
C. pseudobrachyspora	CPC 28808 T	MF490819	MF490841	MF490862
C. pseudolunata	UTHSC 09-2092 T	HE861842	HF565459	–
C. radicicola	CGMCC3.19327 T	MN215699	MN264131	MN263992
C. radici-foliigena	CGMCC 3.19328 T	MN215695	MN264127	MN263988
C. radici-foliigena	LC11954	MN215696	MN264128	MN263989
C. ravenelii	BRIP 13165 T	JN192386	JN600978	JN601024
C. senegalensis	CBS 149.71	HG779001	HG779128	–
C. spicifera	CBS 274.52	JN192387	JN600979	JN601023
C. tamilnaduensis	SZMC 22226 T	MN628311	MN628307	MN628303
C. tamilnaduensis	SZMC 26758	MN628308	MN628304	MN628300
C. tamilnaduensis	SZMC 26759	MN628309	MN628305	MN628301
C. tripogonis	BRIP 12375 T	JN192388	JN600980	JN601025
C. tuberculata	CBS 146.63 IsoT	JX256433	JX276445	JX266599
C. warraberensis	BRIP 14817 T	MH414909	MH433653	MH433672
Bipolaris peregianensis	BRIP 12790 T	JN601034	JN600977	JN601022
Note: ‘–’ indicates that there are no EF-1α genes in the GenBank accession, T = Ex-type.

Koch’s Postulate test

The conidia harvested from 2-week-old cultures on water agar (WA) were resuspended to make conidial suspension (10⁶ spores/ml) with sterile distilled water. In each experiment, six healthy C. kwangsiensi seedlings growing for 2 weeks were used for pathogenicity assay. After surface disinfection with 75% ethanol, 3 were sprayed with 2 mL conidial suspension of the representative strain, and the other 3 were treated with 2 mL sterile water as control(Wei et al. 2023). All the inoculated plants were kept in a greenhouse at 25°C with 12 h photoperiod and approximately 90% relative humidity. Koch's postulates were confirmed by re-isolating the disease pathogen from plants which had been inoculated and infected using the tissue separation technology described above.

Laboratory Fungicides Screening

The fungicidal activities of mancozeb (content: 70%), difenoconazole (content: 10%), trifloxystrobin·tebuconazole (content: 75%), triadimefon (content: 15%) and thiophanate-methyl (content: 50%), which were mainly registered to control fungal disease, were determined via a mycelial growth rate method in vitro. In order to obtain different concentrations (Table 3), each fungicide was dissolved in sterile distilled water and added to the PDA medium. Each treatment was replicated three times. Mycelial disks (5 mm diameter) from the edge of the colonies were transferred onto the center of PDA plates and incubated in an incubator at 28°C. After 5 days, colony diameter was measured using a ruler and inhibition rate I (%) was calculated by the following formula, where C (cm) and T (cm) represent the fungi diameters of the control and treated PDA plates, respectively(Vincent 1947; Hui et al. 2022). Meanwhile, the EC50 (concentration for 50% of maximal effect) values of mancozeb, difenoconazole, trifloxystrobin·tebuconazole, triadimefon and thiophanate-methyl against CK21.1 were calculated using the Graph Pad Prism software.

Table 3

The concentrations of fungicides used in this study.
Fungicides	Concentrations (mg/L)
Mancozeb 70% WG	0.63, 1.25, 2.50, 5.00, 10.0
Difenoconazole 10% WG	0.125, 0.25, 0.50, 5.00, 50.0
Trifloxystrobin·Tebuconazole 75% WG	0.125, 0.25, 0.50, 5.00, 50.0
Triadimefon 15% WP	6.25, 12.5, 25.0, 50.0, 100.0
Thiophanate-methyl 50% WP	15.0, 20.0, 30.0, 40.0, 50.0
Note: WP, wettable powder; WG, water-dispersible granules.

Inhibition rate I (%) = (C - T) / (C − 0.5) × 100.

Phylogenetic analyses

A phylogenetic tree included 47 reference isolates, was generated from the combined dataset of ITS, GAPDH, and EF-1α sequences, and Bipolaris peregianensis BRIP 12790 was used as the outgroup. A total of 2102 characters, including gaps (579 for ITS, 557 for GAPDH, and 966 for EF-1α), were included in the phylogenetic analysis. The tree topologies from both ML and BI analyses were nearly identical (Fig. 1). The phylogenetic tree showed that strains CK21.1, CK21.6, CK21.9, CK21.14 and CK21.18 from this study formed into a distinguished clade from the other known Curvularia species. And the tree also showed that the strains were closely related with C. tamilnaduensis, C. radici-foliigena, and C. radicicola.

The sequences derived from the strain CK21.1 were submitted to the GenBank database. The obtained ITS (OR575881), GAPDH (OR576905), and EF-1α (OR576900) sequences showed 100% (520/520 bp), 99.75% (407/408 bp), and 100% (848/848 bp) identity with the ex-type strain C. tamilnaduensis SZMC 22226 (MN628311, MN628307, MN628303, respectively); 100% (521/521 bp), 99.9% (542/545 bp), and 100% (926/926 bp) identity with the ex-type strain C. radici-foliigena CGMCC 3.19328 (MN215695, MN264127, MN263988, respectively); 100% (516/516 bp), 97.81% (535/547 bp), and 100% (926/926 bp) identity with the ex-type strain C. radicicola CGMCC3.19327 (MN215699, MN264131, MN263992, respectively); 99.88% (528/534 bp), 98.74% (391/396 bp), and 99.28% (830/836 bp) identity with the ex-type strain C. coimbatorensis SZMC 22225 (MN628310, MN628306, MN628302, respectively). The ITS and EF-1α BLAST searches are of limited value in identifying pathogenic organisms, only the GAPDH gene sequence showed differences with the ex-type strain sequences of C. tamilnaduensis, C. radici-foliigena, and C. radicicola.

Taxonomy

Curvularia lingshanensis H. Zhou & H.Y. Wang, sp. nov. (Fig. 2)

MycoBank accession number:849955

Etymology. Name refers to the county in Lingshan, Guangxi Province where the strain was isolated.

Type. CHINA, Guangxi Province, Lingshan County, on Curcuma kwangsiensis, May. 2021.

Description. Colonies on PDA fast growing, with mycelial growth rate = 16.8 ± 0.25 mm/d at 28°C, medium sparse, thinly hairy, smooth surface, well defined margins; from above, the colony appears smooth, sparse, grey; from below, it appears olive-green; Colonies on OA with mycelial growth rate = 16.0 ± 0.29 mm/d at 28°C, round with a complete edge, cotton, a clearly defined margin, and dense wool; Colony with smooth surfaces, green fruiting zone, pale gray aging zone; dark green from below; Colonies on MEA slower growth than PDA and OA, with mycelial growth rate = 12.5 ± 0.32 mm/d, irregular edges, wavy, raised; colony from above, whitish in the production zone, brown in the fruiting zone, and grey in the ageing zone; from below, light gray in the fruiting zone, light brown in the productive zone, and dark brown in the ageing zone.

Hyphae subhyaline septate, branched, smooth, 1.5 ~ 4 µm diameter. Conidiophores erect, short, arising singly, septate, usually unbranched, cell wall thicker than vegetative hyphae, uniformly brown size 11.5 ~ 35 × 2.7 ~ 4.9 µm (average ± SD: = 23.4 ± 6.2 × 3.8 ± 0.5 µm, n = 50). Conidiogenous cells proliferating terminal, sympodially, slightly flexuous, slightly verruculose to smooth-walled, light brown, size 3.2 ~ 12.0 × 2.5 ~ 5.3 µm (average ± SD: 6.9 ± 1.8 × 4.0 ± 0.6 µm, n = 40) Conidia straight, clavate, seldom curved at the third cell from the base, most with 3 septate, and a few 2 septate, olivious brown, size 11.0 ~ 27.9 × 4.5 ~ 11.0 µm (average ± SD: 18.9 ± 3.4 × 7.3 ± 1.1 µm, n = 70). Chlamydospores not observed. Hilum thick-ened, darkened.

Notes

Curvularia lingshanensis is phylogenetically most closely related to C. tamilnaduensis, C. radici-foliigena, C. radicicola, C. coimbatorensis and C. petersonii (Fig. 1), but differs in length of conidiophores (e. g. 11.5 ~ 35 × 2.7 ~ 4.9 µm in C. lingshanensis vs. 125 × 2.5 ~ 4.0 µm in C. tamilnaduensis; 70 ~ 135 × 3.5 ~ 5 µm in C. radici-foliigena; 90 ~ 155 × 4.5 ~ 5.5 µm in C. radicicola; 210 × 3 ~ 4 µm in C. coimbatorensis and 110 ×4 µm in C. petersonii). And differs in conidiogenous cells characteristic (e. g. terminal in C. lingshanensis vs. terminal or intercalary in other species). Furthermore, C. lingshanensis can also be distinguished by the shaped and sized conidia (Table 4).

Table 4

The microscopic characteristics of the new species of *Curvularia* and their relatives proposed.
Specie	Isolate	Conidiophores		Conidiogenous cells		Conidia		References
Specie	Isolate	Characteristic	Size (µm )	Characteristic	Size (µm )	Shape	Size (µm )	References
C. lingshan ensis	CK21	E, S, P, U, B	11.5 ~ 35×2.7 ~ 4.9	T, F, M to V, PB	3.2 ~ 12×2.5 ~ 5.3	H, C, P(2 ~ 3), OB	11 ~ 27.9×4.5 ~ 11	This study
C. tamilnadu ensis	SZMC 22226	E or F, P, U, B	125×2.5 ~ 4	T or I, M, PB to B	–	A to C to X	(15-)20 ~ 23(-28)×(7-)8 ~ 10(-11)	(Kiss et al. 2019)
C. radici-fol iigena	CGMCC 3.19328	E or F, L, P, R, PB	70 ~ 135×3.5 ~ 5	T or I, M to V	5 ~ 9.5×3 ~ 5	H, X, P(2 ~ 3), PB to OB	10 ~ 16.5×5.5 ~ 8.5	(Raza et al. 2019b)
C. radicicola	CGMCC 3.19327	F, L, R, DB	90 ~ 155× 4.5 ~ 5.5	T or I, M, PB to DB	3.5 ~ 18× 2.5 ~ 12	IC, X, P(3), B to DB	6 ~ 18×5 ~ 10.5	(Raza et al. 2019b)
C. coimbator ensis	SZMC 22225	E or F, P, U, B	210×3 ~ 4	T or I, M, B	–	A to C to X	(13-)16–18(-23)× (7-)8–9(-10)	(Kiss et al. 2019)
C. petersonii	BRIP 14642	E or F, P, U or R, B	110 × 4	T or I, M, PB to B	–	H to IC, A to X, P(3), B	(15–)17 ~ 19(–21)× (5–) 5.5 ~ 6 (–7)	(Pei et al. 2018)
Notes: A = Ellipsoidal; B = Brown; DB = Dark brown; OB = Olivious brown; PB = Pale brown; C = Clavate; IC = Slightly curved; E = Erect; F = Slightly flexuous; H = Straight; I = Intercalary; L = Long; M = Smooth; P = Septate; R = Branched; S = Short; T = Terminal; U = Unbranched; V = Verruculose; X = Obovoid.
‘–’ indicates that there are not mentioned in the references.

Pathogenicity test

In May 2021, a stem and leaf rot disease in C. kwangsiensi was found in Lingshan County, Guangxi Province. During the early stages of infection, brown, water-soaked, irregular lesions appeared on tender leaves on the top of the stems. With the development of the disease, lesions extended onto the entire tender leaves and stems, lower leaves could also be affected. Finally, the entire plant turned yellow, soft, slimy, leading to death (Fig. 3). In this study, 21 isolates showed morphological similarities to the genus Curvularia were isolated from three symptomatic plants. Six isolates chosen as representative, were taken for the follow-up study.

Pathogenicity test results showed that the strain CK21.1 can cause stem and leaf rot disease on C. kwangsiensi. Healthy C. kwangsiensi seedlings sprayed with CK21.1 conidial suspension showed rot symptoms 6 days post inoculation. After 12 days, the stems of the plants were completely corrupt and the base of the leaves also showed water-soaked necrosis (Fig. 3). The symptoms were similar to the disease on C. kwangsiensi in the field. No symptoms were detected in the control groups. To fulfill Koch’s postulates, C. kwangsiensi was consistently re-isolated from the necrosis of all inoculated plants and identified by morphology and DNA sequencing, while no isolates were isolated from the negative control plants.

Screening of Fungicides Analyses

In order to screen active fungicides against the pathogen, in present study, the toxicity of the five fungicides, including mancozeb, difenoconazole, trifloxystrobin·tebuconazole, triadimefon and thiophanate-methyl, on the isolate CK21.1 were determined on PAD. The results are shown in Table 5 and Fig. 4. There was a high correlation between fungicide dosage and CK21.1 inhibition for all five fungicides, with correlation coefficients above 0.90. And the antifungal activity was stronger with the increase of the concentration. Furthermore, mancozeb, difenoconazole, and trifloxystrobin tebuconazole showed the best inhibitory effects on CK21.1, with EC₅₀ values of 1.5233 ± 0.18 mg/L, 0.1748 ± 0.03 mg/L, and 0.2557 ± 0.06 mg/L, respectively. These were followed by triadimefon, with EC₅₀ values of 10.3093 ± 0.53 mg/L. In contrast, thiophanate-methyl had the lowest antifungal activity against the pathogen, with an EC₅₀ value of 55.7213 ± 0.68 mg/L.

Table 5

Regression equation and EC₅₀ value of fungicides against the *C. lingshanensis* sp. nov.
Fungicides	Regression equation	Relativity	EC₅₀ (mg/L)
Mancozeb 70% WG	y = 1.8270x + 5.271	0.9575	1.5233 ± 0.18 c
Difenoconazole 10% WG	y = 0.5613x + 5.092	0.9117	0.1748 ± 0.03 c
Trifloxystrobin·Tebuconazole 75% WG	y = 0.4499x + 5.196	0.9502	0.2557 ± 0.06 c
Triadimefon 15% WP	y = 0.9751x + 3.408	0.9668	10.3093 ± 0.53 b
Thiophanate-methyl 50% WP	y = 2.7150x + 0.259	0.9727	55.7213 ± 0.68 a
Note: Different lowercase letters (a-c) significant levels of difference at P < 0.05.

In present study, phylogenetic analysis based on a combined dataset (ITS, GAPDH and EF-1α) indicated that the strains from Curcuma kwangsiensi were related to Curvularia tamilnaduensis N. Kiss & S. Kocsubé, Curvularia radici-foliigena M. R aza, K.D. Hyde & L. Cai, and Curvularia radicicola M. Raza, K.D. Hyde & L. Cai. DNA sequences from the regions of the genes ITS, GAPDH and EF-1α were compared. In the ITS and EF-1α gene regions, for C. tamilnaduensis, C. radici-foliigena, C. radicicola, the base sequences were identical to corresponding DNA sequences of the isolates. In the GAPDH gene region, only 1 difference for C. tamilnaduensis; base differences were 3 for C. radici-foliigena and 12 for C. radicicola. The results suggested that GAPDH has evolved faster than ITS and EF-1α in Curvularia, which is similar to what has been reported (Zhang et al. 2020). Furthermore, the new species can be morphologically differentiated mainly by micromorphology(Qian et al. 2020). The main morphological characteristics that distinguish Curvularia lingshanensis from related species are the length of conidiophores, the shape of conidia, and the characteristics of conidiogenous cells. Conidiophores of C. lingshanensis were shorter than those of C. tamilnaduensis (125 µm)(Kiss et al. 2019), C. radici-foliigena (70 ~ 135 µm)(Raza et al. 2019a)d radicicola (90 ~ 155 µm)(Raza et al. 2019a). The conidiogenous cells of C. lingshanensis were terminal, but the other related species were terminal or intercalary. For C. tamilnaduensis, the conidia were ellipsoidal to obovoid, while these characteristics are not present in C. lingshanensis.

The pathogenicity test showed that the stems of the infected seedlings showed a watery stain, and as the infection time increased, the stems and leaf bases began to decay, and eventually death. To our knowledge, this is the first report of stem and leaf rot disease of C. kwangsiensi caused by Curvularia genus worldwide. Curvularia is a cosmopolitan genus that includes pathogens of a variety of plant hosts(Marin-Felix et al. 2020). Curvularia disease is one of the common fungal diseases. C. lunata infection causes leaf spot on maize(Shaoqing et al. 2022) and root rot on Strawberry(S and K 2010). C. eragrostidis can cause leaf spot on Sansevieria trifasciata(Kee et al. 2020) and postharvest rot on Pineapple(S et al. 2014), C. hawaiiensis infection causes leaf spot on rice(Aslam et al. 2019). As the new Curvularia species, C. lingshanensis should be studied for biological characteristics and host range.

At present, chemical fungicide plays an important role in the control of plant fungal diseases. Difenoconazole belongs to the triazole fungicide, with high efficiency, broad spectrum, safety, and long duration, often used to prevent and control a variety of fungal diseases, and is friendly to the environment(Zhangliang et al. 2023). Mancozeb is a dithiocarbamate non-systemic fungicide acting in a multi-site mode, widely used for the fungal pathogens control in several crops, including tree fruits, field crops, ornamental plants, and sod farms. Mancozeb carries out the protective activity on leaf surfaces and does not penetrate through the cuticle(Gullino et al. 2010).Trifloxystrobin exhibits a high activity against the diseases caused by the memebers of the Ascomycete, Deuteromycete, Basidiomycete, and Oomycete classes, by targeting cytochrome bc1 in mitochondria to inhibit respiration in fungal cells. Fungicide tebuconazole is an ergosterol biosynthesis inhibitor(Mohapatra 2014; Sharma et al. 2019). In this study, the bioassay test results indicated that all the five tested fungicides had antifungal effects against the pathogen. The 70% mancozeb, 75% trifloxystrobin tebuconazole and 10% difenoconazole had the strongest inhibitory effect on C. lingshanensis with the EC₅₀ values of 1.5233 ± 0.18 mg/L, 0.2557 ± 0.06 mg/L and 0.1748 ± 0.03 mg/L, respectively.

This study may provide a theoretical basis for effectively controlling the stem and leaf rot disease of C. kwangsiensi. As laboratory bioassay results are not always accurate reflections of control efficacies in the field, field trials are necessary.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 32060599), and the Natural Science Foundation of Guangxi Zhuang Autonomous Region (grant no. 2021GXNSFAA075023).

Author Contributions

Conceptualization and methodology, H.W. and H. Z.; validation and formal analysis, H.W. and J. S.; Investigation, H.W., Z. L., S. L., and J. G.; writing—original draft preparation, H. W.; writing—review and editing, H. Z.; project administration and funding acquisition, Q. G. and H. Z. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflict of interest.

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Curvularia lingshanensis sp. nov., a New Pathogen Causing stem and leaf rot on Curcuma kwangsiensis and Its Sensitivity to Five Fungicides

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Abstract

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Introduction

Materials and methods

Isolation

Morphological studies

DNA Extraction and Sequencing

Koch’s Postulate test

Laboratory Fungicides Screening

Results

Phylogenetic analyses

Taxonomy

Pathogenicity test

Screening of Fungicides Analyses

Discussion

Declarations

References

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