Isolation, Characterization and Phylogenetic Analysis of Stagonospora tainanensis, the Pathogen Causing Sugarcane Leaf Blight in China

Abstract

Sugarcane leaf blight (SLB), a major fungal leaf disease of sugarcane (Saccharum spp.), has been attributed to Stagonospora tainanensis. In December 2020 and May 2021, signs of leaf blight were observed on sugarcane in the fields of Chongzuo City, in the Guangxi Province of China. Lesions on the leaves were characterized by yellow or dark red spots in the center. Fungal species were isolated, purified and subjected to pathogenicity evaluation on the sugarcane plants. An isolate that caused symptoms the same as those observed in the field was initially identified as S. tainanensis (Leptosphaeria taiwanensis, perfect state) based on its morphological characteristics both of asexual and sexual stages. Dark brown and nearly spherical pycnidia with conidia of long ellipsoidal, hyaline, one to four cells and 29.27 to 54.39 μm long and 9.03 to 16.12 μm wide were found on corn meal agar medium. Ascomata with asci of cylindrical to clavate, a short stipe and eight spores slightly constricted at the septum, with the size of the spore ranging from 36 to 44 μm long and 8.5 to 12 μm wide, were formed on the sugarcane-leaf-decoction saccharose agar medium. The identity of the species was further confirmed by rDNA ITS and TEF-1α sequencing. The optimal temperature for mycelial growth was 25 °C and the optimal pH was 6.0. The pathogen grew well in a medium with oats as the carbon source and yeast extract as the nitrogen source, but poorly in a medium with urea as the nitrogen source. This study is the first to identify the sugarcane leaf blight pathogen in Guangxi, and the first publication describing the biological characterization of S. tainanensis. The occurrence of sugarcane leaf blight should alert sugarcane breeders and plant pathologists to consider integrating control of this potentially important disease into the agenda of their breeding and disease control programs.

1. Introduction

Sugarcane (Saccharum spp.), a monocotyledonous plant in the family Gramineae, is one of the most important sugar-producing crops, accounting for approximately 80% of the world’s total sugar production. It is also a bio-energy plant and serves as silage for livestock [1,2,3]. In China, sucrose from sugarcane accounts for more than 90% of the total sugar consumed [4]. As a globally important cash crop, sugarcane is widely planted in tropical and subtropical regions. The sugarcane industry constitutes an economic pillar in many developing countries to fight against poverty and to improve people’s livelihood [5]. In addition to sugar, sugarcane by-products provide raw materials for other industries [6]. As a perennial crop, vegetative propagation promotes the accumulation of pathogens, which affects the growth and metabolism of the sugarcane plants, resulting in the deterioration of the crop, and thereby causing a decreased yield and lowered resistance [7,8]. The incidence of sugarcane diseases is increasing at an alarming rate, and a considerable portion of sugar production is annually lost due to disease [9].

Sugarcane leaf blight (SLB), caused by the fungal pathogen Stagonospora tainanensis, is a leaf disease of sugarcane, causing leaf blight symptoms and rapid leaf senescence [10]. The disease was reportedly epidemic in Taiwan of China in 1976 when over 10,000 hectares of sugarcane were infected [11]. The disease has been prevalent in the main sugarcane planting areas of China in recent years in Guangxi, Yunnan, Guangdong and Fujian provinces, resulting in severe yield loss [12]. Based on our field survey in Fusui County of Guangxi Province, yield loss could reach 20–30% in susceptible varieties.

S. tainanensis was proposed to be the imperfect state of ascomycete Leptosphaeria taiwanensis when its sexual stage was observed [13]. Despite its relatively long history of discovery, the literature on this pathogen has been limited to descriptions of morphology and taxonomy [14], disease-resistance breeding [11], host resistance [12] and genome assembly [15]. Studies on the biology of the sugarcane leaf blight fungus have been scarce, making it difficult to study the pathogenic mechanism of this fungus on sugarcane.

In December 2020 and May 2021, suspected sugarcane leaf blight was found in a sugarcane field in Chongzuo City, Guangxi Province, China, and the diseased leaves were collected for laboratory identification. In this study, the sugarcane leaf blight pathogen was identified through morphological observation, molecular techniques and pathogenicity tests of the isolates. To understand the epidemic behavior of the pathogen in the wild and to provide a theoretical basis for the prevention and control of the disease, we determined its biological characteristics and thereby identified the relationship between the pathogen’s mycelial growth and temperature, pH, illumination conditions and different nitrogen and carbon sources.

2. Materials and Methods

2.1. Field Survey, Pathogen Isolation and Verification

Sugarcane leaves with leaf blight were collected from a trial plot in Chongzuo City, Guangxi Province of China in December 2020 and May 2021. Tissue sections infected by the causal agent measuring approximately 4 × 4 mm were collected at the junction of healthy and diseased tissue. The leaves were surface-sterilized with 75% ethanol for 15 s and with 0.1% mercuric chloride for 1 min, then transferred into sterile water for 30 s (thrice) for disinfection, followed by incubation in a potato dextrose agar (PDA) culture medium containing 50 μg/mL streptomycin sulfate. The culture vessels were placed upside down in an incubator at a constant temperature of 25 °C for 72 h in the dark.

After a colony had formed around a tissue section, the edge of the mycelium was transferred to a potato dextrose agar (PDA) culture medium containing 50 μg/mL streptomycin sulfate with sterilized toothpicks, and cultured for 48 h. The isolates were purified by successive mass transfer on PDA. The isolates obtained were further purified and stored in 20% glycerol at 4 °C for further studies.

Five-week-old (6th leaf stage) sugarcane plants, which were cultivated from cuttings of the symptom-free cultivar Zhongzhe 9, were used to test the pathogenicity of the isolates. Each strain was cultured on a corn meal agar (CMA) medium (30 g of corn meal, 18 g of agar, 1000 mL of distilled water and 50 μg/mL streptomycin sulfate) for 14 days (25 °C, 24 h with 365 nm near-ultraviolet, NUV). Then, the conidia suspension was spread on a corn meal agar medium for 5–7 days (25 °C, 24 h NUV), 5 mL of distilled water was added, the conidia were scraped with a sterilized cover glasses and filtered with gauze and the concentration was adjusted to 5 × 10⁴ spores/mL. A spore suspension (5 × 10⁴ spores/mL) with 0.1% Tween 80 was sprayed on the leaves of Zhongzhe 9 (3 mL per plant of six leaves). Distilled water containing 0.1% Tween 80 was used as the control. Nine sugarcane plants were inoculated with each treatment. The plants were kept in a glasshouse at 25–28 °C and covered with plastic bags to retain moisture. The plastic bags were removed after 2 d and the plants were observed and photographed at 3, 7, 10, 15 and 30 d. Infected leaves were collected to isolate and morphologically identify the pathogen via Koch’s postulates.

2.2. Morphological Identification

Eleven isolates were selected for comparison and characterization. Edge hyphae from purified colonies were selected and transferred to the center of a potato dextrose agar (PDA) medium (46 g PDA meal, 1000 mL of distilled water and 50 μg/mL streptomycin sulfate), and cultured at 25 °C in the dark for 9 d. To observe the conidial characteristics, these isolates were cultured on a corn meal agar medium, and continuously irradiated with NUV at 25 °C for 14 days to induce the production of mature conidia. At the same time, a representative strain (SF007) was selected for the induction of sexual generation. Isolates were grown on a sugarcane-leaf-decoction saccharose agar (SSA) medium (a filtered decoction of 200 g of fresh sugarcane leaves that had been boiled for 30 min, containing 20 g of sucrose, 20 g of agar, 1000 mL of distilled water and 50 μg/mL of streptomycin sulfate), and continuously irradiated with NUV at 25 °C for 20 days to induce the production of mature ascoma. Samples were sectioned using a model Hestion CM2850 freezing microtome (Hestion Corporation, Changzhou, China). The morphology was observed microscopically using Olympus BX 53 (Olympus Corporation, Tokyo, Japan) and Keyence VHX-6000 (Keyence Corporation, Osaka, Japan). The morphology, color, conidia size and ascospores (100 replicates) were observed, measured and photographed.

2.3. Molecular Studies

The ribosomal internal transcribed spacer (ITS) region and the translation elongation factor-1α (TEF-1α) gene were used for molecular identification. DNA isolation was performed utilizing the Plant Genomic DNA Kit (TransGen Biotech, Beijing, China). Isolates were grown on PDA for 5 days before their DNA was extracted. A polymerase chain reaction (PCR) was performed by using the primers for ITS (ITS1 5′-TCCGTAGGTGAACCTGCGG-3′; ITS4 5′-TCCTCCGCTTATTGATATGC-3′) [16] and TEF-1α (EF-983F 5′-GCYCCYGGHCAYCGTGAYTTYAT-3′; EF-2218R 5′-ATGACACCRACRGCRACRGTYTG-3′) [17] to amplify ITS and TEF-1α, respectively. Each 25 mL amplification reaction system included 1 μL of fungal DNA (50 ng/μL), 1 μL of each primer (10 pmol final concentration of each primer), 9.5 μL of ddH₂O and 12.5 μL of 2 × Rapid Taq MasterMix (Vazyme Biotech, Nanjing, China). The PCR was performed using a ProFlex 3 × 22-Well PCR System (Life Technologies, Singapore) with the following thermal profile: 3 min at 94 °C for initial denaturation, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 30 s (ITS) or 40 s (TEF-1α) at the primer-pair-specific annealing temperature of 54 °C (ITS) or 60 °C (TEF-1α) and extension at 72 °C for 30 s. A final extension was performed at 72 °C for 10 min. The PCR product was separated via electrophoresis on a 1.5% agarose gel running for 20 min in a TBE buffer, then the agarose gel DNA fragment was recovered and purified using the HiPure Gel Pure DNA Mini Kit (Magen Biotech, Guangzhou, China) according to the manufacturer’s protocol. The PCR products were cloned into the pEASY-T1 Cloning Kit Vector (TransGen, China). Three positive transformants with PCR products were sent to a commercial sequencing service provider (Augct Biotech, Wuhan, China) for DNA sequencing. For each gene sequence and isolate, three positive transformants were prepared and sent for DNA sequencing. The sequences were analyzed via the BLAST search program on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi/ (accessed on 1 October 2021)) and submitted to GenBank.

The GenBank accession numbers of the sequences generated in this study are included in

Table 1. Sequence and information of isolates in this study.

Species	Isolate No.	Host	Location	GenBank Accession Number
				ITS	TEF-1α
S. tainanensis	SF001	sugarcane	Chongzuo, Guangxi	ON428231	ON469971
S. tainanensis	SF002	sugarcane	Chongzuo, Guangxi	ON428232	ON469972
S. tainanensis	SF003	sugarcane	Chongzuo, Guangxi	ON428233	ON469973
S. tainanensis	SF004	sugarcane	Chongzuo, Guangxi	ON428234	ON469974
S. tainanensis	SF005	sugarcane	Chongzuo, Guangxi	ON428235	ON469975
S. tainanensis	SF006	sugarcane	Chongzuo, Guangxi	ON428236	ON469976
S. tainanensis	SF007	sugarcane	Chongzuo, Guangxi	ON428237	ON469977
S. tainanensis	SF008	sugarcane	Chongzuo, Guangxi	ON428238	ON469978
S. tainanensis	SF009	sugarcane	Chongzuo, Guangxi	ON428239	ON469979
S. tainanensis	SF010	sugarcane	Chongzuo, Guangxi	ON428240	ON469980
S. tainanensis	SF011	sugarcane	Chongzuo, Guangxi	ON428241	ON469981

. A phylogenetic tree was generated using amino acid sequences of TEF-1α genes, and MEGA 11.0.11 with maximum-likelihood and the Poisson model. Confidence levels of the branching points were determined using 1000 bootstrap replicates.

2.4. Characterization of the Fungus In Vitro Cultural Conditions

The effect of temperature on the mycelial growth was studied by sampling 6 mm mycelial plugs using a sterilized punch from the edges of colonies that had been cultured for 6 days. The mycelial plugs were transferred to the PDA medium and cultured at 5, 15, 20, 25, 28, 30 and 35 °C. The colony diameters were measured with crossing methods after 3, 5, 7 and 9 days of cultivation, and the results for each treatment were presented as the average of three replicates.

Furthermore, the effects of pH and illumination on mycelial growth were analyzed. Mycelial plugs were transferred to the PDA medium and cultured at pH 2, 4, 5, 6, 7, 8, 10 and 12 by using HCl and NaOH to adjust the pH of the PDA medium. Five light conditions were employed, consisting of 24 h of darkness, 24 h of white light, 12 h white light/12 h darkness, 24 h NUV and 12 h NUV/12 h darkness. Inoculation, culture and measurements were performed as described above.

Various nitrogen sources were screened: tryptone, yeast extract, NaNO₃, Casein acid Hydrolysate, KNO₃, beef extract, NH₄NO₃ and urea were used to replace NaNO₃ at the same nitrogen content in a Czapek-Dox agar medium (2 g of NaNO₃, 1 g of K₂HPO₄, 0.5 g of MgSO₄·7H₂O, 0.5 g of KCl, 0.01 g of FeSO₄, 30 g of sucrose, 20 g of agar, 1000 mL of sterile water and 50 μg/mL streptomycin sulfate). A nitrogen-free medium was used as a control. Inoculation, culture and measurements were performed as described above.

Various carbon sources were also screened: corn meal, D-mannitol, glucose, sucrose, maltose, soluble starch, maltodextrin, glycerin and oats were used to replace sucrose at the same carbon content in the Czapek-Dox agar medium. A carbon-free medium was used as a control. Inoculation, culture and measurements were performed as described above.

2.5. Statistical Analysis

All data were statistically analyzed using the SPSS 26.0 software package (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance (ANOVA) and Duncan’s test (p = 0.05) were used to determine the presence of significant differences between treatments. Data are presented as the mean ± SD.

3. Results

3.1. Field Survey and Pathogen Isolation

Sugarcane leaf blight was found at all growth stages of sugarcane leaves in the field in the years of 2020 and 2021 in Fusui County of Guangxi Province, China (Figure 1). The lesions were normally long fusiform, and yellow or red. Dark red infection points occurred in the center of the lesions. In the late stage of infection, the lesions gradually expanded to cover the entire leaf. Eleven isolates (SF001–SF011) were recovered from sugarcane plants with symptoms of leaf blight.

Typical SLB symptoms were observed on Zhongzhe 9 plants inoculated with conidial spores of isolate SF007 (Figure 2), whereas the control remained asymptomatic. The symptom development was in the following sequences: dark red infection spots were first observed on the leaves of the inoculated sugarcane plants 2–3 days after inoculation (Figure 2b); chlorosis extended from the spots longitudinally in both directions (Figure 2c,d); the lesions gradually turned red to dark red by day 15 (Figure 2e); and by day 30, spots merged into a large erythema-like lesion (Figure 2f). Subsequently, the same fungus was successfully re-isolated from the lesions to fulfill the requirements of Koch’s postulates.

3.2. Morphological and Phylogenetic Analysis of the Pathogen

Eleven isolates were selected for further analysis. These isolates grew well when cultured on PDA at 25 °C in complete darkness (Figure 3a,b). After 9 days, the diameter of the colonies differed slightly among isolates (71.00 to 82.33 mm) (

Table 2. Morphological descriptions of the pathogens causing sugarcane leaf blight investigated in this study.

Isolate	Colony Diameter (mm)	Conidial Spore (μm)
		Length	Width
SF001	77.00 ± 0.41	38.13 ± 4.64	12.14 ± 1.06
SF002	79.67 ± 0.47	40.09 ± 2.42	11.89 ± 0.73
SF003	81.83 ± 0.24	40.25 ± 3.72	11.94 ± 1.03
SF004	73.17 ± 0.23	39.98 ± 4.21	11.61 ± 1.22
SF005	80.50 ± 0.24	39.17 ± 3.34	11.75 ± 1.11
SF006	73.33 ± 0.24	41.44 ± 2.84	12.07 ± 1.11
SF007	82.33 ± 0.47	41.40 ± 3.91	11.83 ± 1.15
SF008	71.00 ± 1.08	38.31 ± 3.58	11.52 ± 0.96
SF009	79.17 ± 0.24	41.12 ± 3.26	11.40 ± 0.92
SF010	79.67 ± 0.47	40.42 ± 3.07	11.08 ± 0.77
SF011	80.33 ± 0.47	41.84 ± 2.54	12.04 ± 0.72

), averaging approximately 78.00 mm. Generally, the colonies appeared circular, with dense milky-white or gray-white hyphae on the PDA medium. None of the isolates sporulated when the mycelial plugs were cultured on the PDA medium. However, these isolates produced mature conidia when cultured on a corn meal agar (CMA) medium and induced with 24 h NUV light after 14 days (Figure 3c–e). Nearly spherical, dark brown pycnidia were scattered and embedded over the medium, and partially mature conidia were oozing from the ostiolum (Figure 4a–c). The conidia were long ellipsoidal, hyaline, one to four cells (each cell contained several oil particles of varying sizes) with septate and 29.27 to 54.39 μm long and 9.03 to 16.12 μm wide.

The colony diameter was measured on day 9 and the size of the conidial spore on day 14. Values are means ± SD of three replicates for the colony diameter and of one hundred replicates for the conidial spore.

The isolate designated as SF007 was cultured on the SSA medium, induced with 24 h NUV light and maintained for 20 days to produce mature ascomata (Figure 4d). Nearly spherical, dark brown ascomata were scattered and embedded over the medium. Asci were cylindrical to clavate, with a short stipe, and eight-spored (Figure 4e,f). Ascospores contained oil droplets and were fusiform, slightly curved, septated and slightly constricted at the septum, with an entire sheath, ranging from 36 to 44 μm long and 8.5 to 12 μm wide (Figure 4g). The conidia and ascospore morphological characteristics of isolate SF007 matched those described by Hsieh [13].

The ITS and TEF-1α regions of the isolates were amplified using the PCR with ITS 1 or ITS 4 [16] and EF-983F or EF-2218R [17] primers and sequenced. The ITS (510 bp) and TEF-1α (1033 bp) sequences of the 11 isolates and alignment analysis showed that the ITS and TEF-1α regions of the isolates were with 99.8% and 100% identities to those of the S. tainanensis (AB808556.1). The phylogenetic tree constructed with the TEF-1α amino acid sequence showed that isolate SF007 formed a clade with S. tainanensis, which distinguished them from other Stagonospora species (Figure 5). Combined with the morphological identification, we concluded that SF isolates all belonged to S. tainanensis.

3.3. Optimized Culture Conditions for the Pathogen

The fungus could grow from 5 °C to 35 °C; the mycelia grew well from 25 °C and 28 °C, yielding colony diameters on day 9 of 81.17 mm and 80.83 mm, respectively. Mycelial growth was inhibited when the temperature was lower than 15 °C or higher than 35 °C. Extreme temperature had a large effect on mycelial growth: there was poor growth at 35 °C and 5 °C and the mycelia ceased to grow at 40 °C (Figure 6A).

Isolate SF007 showed a strong ability to adapt to pH and grew in conditions from pH 2 to pH 12, but grew slowly at either extreme. The fungus showed maximal growth at pH 6 (the colony diameter on day 9 was 78.33 mm), and no significant difference in growth was evident at pH 6, 7 or 8. The fungus preferred a slightly acidic to neutral environment and proved adaptable to extreme pH (Figure 6B).

The fungus grew well under illumination conditions of 24 h darkness, 12 h NUV, 24 h NUV, 12 h light and 24 h light. Under 24 h darkness and 12 h light, the colony was white and grew luxuriously. Under 24 h light, 12 h NUV and 24 h NUV, the center of the colony showed a gray, depressed area, and the depressions were largest under the 24 h of NUV.

As shown in Figure 6C, the fungus grew with all tested carbon and nitrogen sources, but the mycelial growth rates were notably different. The medium with oats as the carbon source had the fastest growth rate (the colony diameter on day 9 was 69.83 mm), followed by media with corn meal and soluble starch as the carbon sources. In this study, the mycelial growth rate of all experimental groups was higher (to varying degrees) than that of the control group (i.e., the medium without a carbon source). In the medium with oats as the carbon source, the colony was gray with dense mycelia. The mycelia grew sparsely on the medium with soluble starch or glucose as carbon sources.

We tested the nitrogen utility by the fungus. In the medium with yeast extract as the nitrogen source, the colony growth rate was the fastest (the colony diameter was 78.83 mm after 9 days of culture). This was followed by the media with beef extract, acid hydrolyzed casein, NH₄NO₃ and tryptone as nitrogen sources; in all these media, the colony growth rates were significantly higher than that of the control group (without nitrogen). The colony growth rate was the lowest in the medium with urea as a nitrogen source. In the medium with yeast extract and beef extract as nitrogen sources, the mycelia grew thickly, and the colonies were white and regular in shape. The mycelia were sparse in the medium with acid-hydrolyzed casein as the nitrogen source. Therefore, oats were the most suitable carbon source for mycelial growth, and yeast extract was the best nitrogen source for the growth of this fungus (Figure 6D).

4. Discussion

In this report, we presented data to prove that S. tainanensis was the causative agent responsible for leaf bright disease on sugarcane in Guangxi of China. Previously, ascospores were reported to be able to initiate infection on sugarcane [13]. We found that conidial spores were also efficient in inciting SLB by artificial inoculation (Figure 2). This finding is consistent with the rapid spread of SLB in the growing season of the sugarcane in the field.

Nutrition seems to play an essential role in the regulation of mycelial growth and conidiation in S. tainanensis, i.e., the fungus grew fastest in the medium with oats as a carbon source, followed by corn meal, and slowest with sucrose (Figure 6C); the fungus could not yield any conidia on PDA but could produce abundant conidial spores on CMA (Figure 3). Similar phenomena have been reported in other phytopathogenic fungi, e.g., Ternstroemia gymnanthera Dieback fungus Neofusicoccum parvum [18]. It is now known that amino acids [19], vitamins [20] and light [21] are among the factors to trigger the process of sporulation in fungi. Nevertheless, the successful induction of conidiation in S. tainanensis offers a powerful means for manipulation of this fungus at a molecular level in the future.

Although S. tainanensis could grow at a temperature range of 5 °C to 40 °C, the optimal temperature for mycelial growth was 25 to 28 °C during in vitro cultivation (Figure 6A). The temperature range of May, June, October and November was 20–30 °C in sugarcane growing regions in Guangxi Province, coinciding with the two epidemic seasons of SLB in the field. Light types showed specific effects on the growth of the pathogen. Compared with other treatments, white light produced more aerial mycelia.

A field survey identified highly resistant and highly susceptible sugarcane varieties, suggesting that there are resistance resources for the disease in the current collection of sugarcane germplasm. Therefore, SLB should be given priority in the future sugarcane breeding program.

5. Conclusions

S. tainanensis was confirmed as the agent causing the leaf blight disease on sugarcane in Guangxi of China by biological and molecular evidence. S. tainanensis could grow on a variety of media in vitro, but preferred oat meal and yeast extract as the carbon and nitrogen sources. CMA could be used to induce conidiation for S. tainanensis.