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Article

Identification and Characterization of Nigrospora Species and a Novel Species, Nigrospora anhuiensis, Causing Black Leaf Spot on Rice and Wild Rice in the Anhui Province of China

by
Yang Liu
1,†,
Jiahao An
1,†,
Asma Safdar
2,
Yang Shen
1,
Yang Sun
1,
Wenhui Shu
1,
Xiaojuan Tan
1,
Bo Zhu
1,
Jiaxin Xiao
1,
Jan Schirawski
3,
Feng He
1,3,* and
Guoping Zhu
1,*
1
College of Life Sciences, Anhui Normal University, Wuhu 241000, China
2
Department of Plant Pathology, College of Agriculture, University of Sargodha, Sargodha 40100, Pakistan
3
Department of Genetics, Matthias Schleiden Institute, Friedrich Schiller University Jena, 07743 Jena, Germany
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2024, 10(2), 156; https://doi.org/10.3390/jof10020156
Submission received: 17 December 2023 / Revised: 9 February 2024 / Accepted: 11 February 2024 / Published: 16 February 2024
(This article belongs to the Special Issue Interactions between Filamentous Fungal Pathogens and Hosts: 2nd Edition)
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Abstract

:
Rice production in the Anhui province is threatened by fungal diseases. We obtained twenty-five fungal isolates from rice and wild rice leaves showing leaf spot disease collected along the Yangtze River. A phylogenetic analysis based on internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF1-α), and beta tubulin (TUB2) sequences revealed one isolate (SS-2-JB-1B) grouped with Nigrospora sphaerica, one (QY) with Nigrospora chinensis, twenty-two with Nigrospora oryzae, and one isolate (QY-2) grouped in its own clade, which are related to but clearly different from N. oryzae. Nineteen tested isolates, including sixteen strains from the N. oryzae clade and the three isolates of the other three clades, caused disease on detached rice leaves. The three isolates that did not belong to N. oryzae were also able to cause disease in rice seedlings, suggesting that they were rice pathogens. Isolate QY-2 differed from the other isolates in terms of colony morphology, cell size, and susceptibility to fungicides, indicating that this isolate represents a new species that we named Nigrospora anhuiensis. Our analysis showed that N. sphaerica, N. chinensis, and the new species, N. anhuiensis, can cause rice leaf spot disease in the field. This research provides new knowledge for understanding rice leaf spot disease.

1. Introduction

Nigrospora species are ascomycetes and include both endophytic and parasitic fungi that are becoming famous [1,2,3,4]. This group of fungi can infect a wide range of plants, thereby causing great economic losses in crop production [2,3,5,6,7,8,9]. Nigrospora species are known to infect Dioscorea opposita [10], Oryza satiwa [11], Chrysanthemum moratorium [3], Passiflora edulis Sims [12,13], Arachis hypogaea [14], Nicotiana tabacum [15], and Photinia serrulata [16] in China and lead to leaf spot or blight symptoms. They seem to have a broad host range. The known rice pathogen Nigrospora oryzae, for example, can also infect yam, wild rice, poplar, kiwifruit, red elephant grass, cotton, and pear [3,8,10,11,17,18,19,20]. In light of the global temperature increase, Nigrospora species will likely cause numerous plant diseases. Thus, the threat caused by Nigrospora species should be drawing our attention, and more knowledge about these pathogens should be gained.
Initially, the characterization of Nigrospora species was based on morphological features, and species identification was performed via a sequence comparison of the internal transcribed spacer (ITS) region [2,5,8,21]. However, among the members of this species, there are very few morphological differences that are limited to differences in colony color, spore size, and spore color [21]. Recently, multi-locus sequence analysis was used for the identification of fungal species. According to an analysis based on a multi-locus phylogenetic tree that was constructed using the combined ITS, TEF1-α, and TUB2 sequences, Nigrospora species were divided into sixteen groups, including the famous rice pathogen N. oryzae, and two unclear groups [2,5,6,10,21]. This analysis also led to the identification of many new species [9,22]. Therefore, species identification based on the multi-locus sequence analysis is more reliable and will be especially beneficial for the characterization of Nigrospora species lacking special morphological features.
N. oryzae, Nigrospora panici, and Nigrospora chinensis are all known to inhabit rice leaves as endophytes [23]. However, N. oryzae is also known as a rice pathogen that causes yellow-to-brown, oval, or circular lesions with large yellow halos on rice leaves [11] and is universally distributed on various plants or dead plant residues in the field. Initially, many of the Nigrospora species were classified as N. oryzae because of the high similarity of their ITS sequences [23]. Therefore, many identified isolates of N. oryzae are probably not completely accurately classified. The correct species identification of rice pathogens is a prerequisite for the successful development of novel rice disease-control strategies.
The provinces along the Yangtze River are important rice production regions in China. Especially in the Anhui province, where the weather is suitable for fungal growth and transmission, there is an abundant fungal biodiversity. Recently, Nigrospora species were discovered to cause leaf spot disease on rice [11]. However, there is little known about this species in the cities along the Yangtze River in the Anhui province. In this study, we investigated rice and wild rice leaf spot diseases in six cities of the Anhui province. Following the collection of diseased rice samples with oval or circular lesions, a molecular analysis was performed. This revealed the discovery of three novel rice pathogens in addition to the known N. oryzae: Nigrospora sphaerica, N. chinensis, and a new species, Nigrospora anhuiensis.

2. Materials and Methods

2.1. Sample Collection of Diseased Leaves and Pathogen Isolation

For sample collection, we screened at least five rice fields each (each field > 1000 acres) at 17 sites near different cities in the Anhui province along the Yangtze River during the rice production season from June to October 2021. Instances of rice leaf spot disease were recorded, and samples of diseased rice leaves were collected. We also screened and sampled the wild rice and grasses near the rice fields. To isolate fungal plant pathogens, collected detached leaves were surface-sterilized by rinsing in 75% ethanol for 30 s and 0.5% sodium hypochlorite for 5 min, followed by three rinses in sterile distilled water, their drying, cutting in 0.5 cm strips, and placement on potato dextrose agar (PDA) containing kanamycin (50 mg/L). Individual isolates were sub-cultured three times on kanamycin-containing PDA. Fungal morphology was recorded by photography after two and four days of incubation at 25 °C.

2.2. Phylogenetic Analysis of Fungal Isolates

Mycelia of fungal isolates were scraped off from PDA plates that were incubated for 2 days at 25 °C. The material was collected and used for genomic DNA isolation [24]. The ITS, TEF1-α, and TUB2 sequences of the isolates were amplified using primer pairs according to a previous study [23]. PCR products were sent for sequencing by the Sangon Company (Shanghai). The newly generated sequences were complemented with the ITS, TEF1-α, and TUB2 sequences from different Nigrospora species available in the NCBI nucleotide database [21], and accession numbers for each gene are presented in Table 1. Nonsense sequences were removed from 5′ and 3′ flanks prior to sequence alignment using MEGA7.0 [25]. The phylogenetic tree was generated using the neighbor-joining method with 1000 bootstrap replicates with the MEGA7.0 software [25]. ITS, TEF1-α, and TUB2 sequences of the new N. anhuiensis isolates were submitted to the NCBI database (Table 1).

2.3. Virulence Testing of Fungal Isolates

For testing virulence of fungal isolates on rice, the fungal isolates were first cultured on PDA for two days. Agar plugs from fungal colony edges were excised from the plates and positioned on detached leaves of six-week-old rice seedlings or on six-week-old rice seedlings grown under natural conditions in the summer, and the sterile PDA plugs were used as control. The inoculated detached rice leaves were incubated at 25 °C for two days, at which lesions were observed and lesion sizes were measured. Each strain was used to inoculate at least three leaves. Lesion sizes occurring on the inoculated seedlings grown under natural conditions were observed one week post-inoculation. Three seedlings were inoculated per fungal isolate. Data for the lesion length were analyzed using one-way ANOVA through SPSS 25 (SPSS version 25; IBM, Chicago, IL, USA), and the QY-2 isolate was used as the reference for ANOVA analysis.

2.4. Analysis of Morphological Features of the Isolates

To observe morphologic similarities and differences of the isolate QY-2 with N. oryzae, N. sphaerica SS-2JB-1B, or N. chinensis QY, all isolates were freshly grown on potato dextrose agar (PDA) and colony features were observed after incubation for two and four days. To observe the features of individual hyphae, we cultured the strains for 24 h on PDA that was spread on sterile microscopic slides and imaged the hyphae using bright light illumination of a fluorescence inverted microscope (Leica, Wetzlar, Germany). We induced conidia formation of the five isolates by cultivation on carrot agar (200 g/L fresh carrot soup, 15 g/L agar powder) at 25 °C for seven days. Conidia were collected from the agar plates and analyzed using microscopy (Leica, Wetzlar, Germany). Of each species, ten conidia were measured in length, and the data were analyzed using Prism GraphPad 8.0 [26].

2.5. Sensitivity Testing of N. anhuiensis and N. oryzae to Eight Different Fungicides

To test sensitivity of N. anhuiensis QY-2 to commercial fungicides, we selected the eight fungicides, chlorothalonil, carbendazim, thifluzamide, flusilazole, thiophanate–methyl, mancozeb, difenoconazole, and prochloraz (Meilinxuehai, Nanjing, China), that are commonly applied in the field for fungal disease control. The QY-2 strain was cultured on the plates with different concentrations (0, 2.5, 5, and 10 µg/mL) of each fungicide, and N. oryzae isolate SS2-1A was used as control. Colony diameters for each plate were measured and photographed at two days of incubation at 25 °C. For each concentration, three technical replicates were prepared. Colony diameters were calculated using the formula Y = (Dn − D0) × 100/D0, where D0 indicates the colony diameter on PDA, and Dn denotes the colony diameter on fungicide containing PDA and analyzed using one-way ANOVA through SPSS 25 (SPSS version 25; IBM, Chicago, IL, USA).

3. Results

3.1. Nigrospora Species Are Associated with Leaf Spot Disease in the Anhui Province, China

The Anhui province is one of the major rice-producing areas in China, located in the eastern part of the Yangtze River basin. To investigate incidences of rice disease in the Anhui province, we screened a total area of more than 17,000 acres of rice fields and neighboring wild rice and grass plants. Incidences of rice disease in the form of circular or oval lesions on rice leaves were widely observed in Qingyang county, Nanling county, and Guichi district, and a few incidences were found in Tongling City and Xuancheng City. Most of the samples were found in private rice fields, where the disease incidence was below 0.1%. In fields managed by companies or institutes, only very few diseased leaves were found. The diseased leaves showed leaf blight and brown-to-black circular, oval, or fusiform lesions, which had a halo of yellow or brown (Figure 1). We isolated and purified leaf-colonizing fungi from diseased rice or wild rice leaves collected at various sites in the Anhui province, China, resulting in a total of 25 novel isolates (Table 2).

3.2. Isolate QY-2 Is a Novel Nigrospora Species Based on Multi-Locus Phylogenetic Analysis

To allow species identification of the novel isolates, we amplified and sequenced their ITS, TEF1-α, and TUB2 genomic regions and used the obtained sequences for multi-locus phylogenetic tree construction after having added available reference sequences of different Nigrospora species. Most (22 of 25) isolates clustered with known N. oryzae isolates and are, thus, identified as N. oryzae isolates (Figure 2; black ovals). One isolate (SS-2JB-1B) was grouped with N. sphaerica isolates (Figure 2; green triangle), one (QY) with N. chinensis isolates (Figure 2; blue triangle), and one isolate (QY-2) was located on a separate branch with a high statistical score that was related to, but clearly different than, N. oryzae (Figure 2; red triangle). Thus, rice disease symptoms in the Anhui province could be associated with fungal isolates of N. oryzae, N. sphaerica, N. chinensis, and a novel Nigrospora spp. that is related to N. oryzae. We named the novel Nigrospora species that is represented by isolate QY-2 N. anhuiensis.

3.3. The New N. anhuiensis Isolate QY-2 Has Similar Morphologic Features as Other Nigrospora Species

We aimed to observe whether the new N. anhuiensis isolate QY-2 could be morphologically differentiated from the well-characterized N. oryzae rice pathogen. For comparison, we included in our analysis the less closely related isolates N. sphaerica SS-2JB-1B and N. chinensis QY. N. anhuiensis QY-2 formed a white colony with few aerial mycelia at 2 days of incubation on nutrient agar, which is similar to that of N. oryzae isolates (Figure 3A). At four days of incubation, the N. anhuiensis QY-2 colony center changed its color to dark green, and the colony formed massive white aerial hyphae (Figure 3A). In contrast, the colony color of the two N. oryzae isolates was palm green or brown, and the massive aerial mycelia formed were grey (Figure 3A). N. sphaerica SS-2JB-1B and N. chinensis QY isolates also produced grey aerial mycelia, but the morphology of the aerial mycelia was different from that of the other three isolates (Figure 3A). All investigated isolates formed smooth, branched, and septate hyphae (Figure 3B). Conidia formation of all isolates could be induced on carrot medium. Conidia of all investigated isolates had a similar globose or oval, black, smooth, and aseptate morphology, while several grey and transparent conidia were also found in QY-2, SS2-1A, and QY isolate (Figure 3C). The sizes of the conidia varied slightly among the isolates. For N. anhuiensis QY-2, conidia diameter was, on average, 12.75 µm but varied by 3 µm between the smallest and the largest measured conidium. Conidia of the N. oryzae isolates were, on average, 12.90 and 13.40 µm but varied only by 1 µm between the smallest and the largest measured conidium (Figure 4). The conidia of N. sphaerica SS-2JB-1B and N. chinensis QY were 13.1 and 13.3 µm on average, with a 1 µm range between the smallest and the largest measured conidium (Figure 4). Based on morphological criteria, N. anhuiensis can best be differentiated from N. oryzae, N. sphaerica, and N. chinensis by its dark green colony center color at four days of incubation on nutrient agar. However, we recommend species identification by multigene sequencing.

3.4. The Identified N. oryzae, N. sphaerica, N. chinensis, and N. anhuiensis Isolates Are Rice Pathogens

To check whether the isolated isolates were rice pathogens, we performed a virulence test on detached rice leaves. Because some of the isolates were collected in the same field and also are N. oryzae isolates, we selected 16 isolates, including most N. oryzae isolates, for virulence testing. A total of 19 of 25 isolates were able to induce black spot lesions on detached rice leaves (Figure 5). This included two isolates that were isolated from wild rice and the identified N. sphaerica, N. chinensis, and N. anhuiensis isolates (Figure 5).
To test whether the isolates were able to cause disease on plants cultivated in the field, we performed a virulence test on rice and wild rice seedlings that were inoculated with either N. oryzae isolates SS2-1A, SS-2-JB-2A, N. sphaerica isolate SS-2JB-1B, N. chinensis isolate QY, or N. anhuiensis isolate QY-2. The inoculated rice seedlings were incubated in natural conditions in the summer. All isolates induced lesions on healthy rice plants. Lesion length was largest with N. anhuiensis isolate QY-2, and N. sphaerica isolate SS-2JB-1B induced the smallest lesions (Figure 6). The lesions caused by N. anhuiensis isolate QY-2 were brown to black and irregular, while the N. sphaerica isolate SS-2JB-1B and the N. chinensis isolate QY caused fusiform lesions with yellow verges (Figure 6). These symptoms were similar to the symptoms in the field. We re-isolated the fungi from the lesions, and the colony features were the same as those of the isolates used for infection. In summary, we conclude that the N. sphaerica, N. chinensis, and N. anhuiensis isolates are the agents that cause rice or wild rice leaf spot disease.

3.5. Sensitivity of N. anhuiensis Isolate QY-2 and N. oryzae Isolate SS2-1A to Various Fungicides

To investigate whether the newly identified rice pathogen N. anhuiensis can be killed by commercial fungicides, we tested the eight fungicides chlorothalonil, carbendazim, thifluzamide, flusilazole, thiophanate–methyl, mancozeb, difenoconazole, and prochloraz that were already registered in China for their capacity to inhibit growth of N. anhuiensis isolate QY-2 and N. oryzae isolate SS2-1A on nutrient agar plates. We observed that all eight tested fungicides could inhibit the mycelial growth of both isolates in a concentration-dependent manner (Figure 7). For most fungicides, N. anhuiensis isolate QY-2 was more sensitive than N. oryzae isolate SS2-1A (Figure 7). Carbendazim was the most effective fungicide against N. anhuiensis isolate QY-2, with a concentration of 5 µg/mL suppressing its growth almost completely (Figure 7). N. anhuiensis isolate QY-2 could still grow on plates containing 10 µg/mL of thifluzamide, flusilazole, mancozeb, and difenoconazole, which suggests that these fungicides might be less effective in eradicating infection with N. anhuiensis in the field (Figure 7). At lower fungicide concentrations, the colony diameter of N. oryzae isolate SS2-1A was dramatically bigger than that of N. anhuiensis isolate QY-2 for the fungicides difenoconazole, chlorothalonil, thiophanate–methyl, and thifluzamide, suggesting that N. oryzae isolate SS2-1A was more resistant to these fungicides (Figure 7). Taken together, these data imply that the N. anhuiensis isolate QY-2 is sensitive to all tested fungicides. However, the growth of the isolate in vitro can be effectively inhibited by carbendazim, which might be the best choice for controlling N. anhuiensis rice spot disease spread in the field.

4. Discussion

This study was conducted to identify the diversity and number of Nigrospora species prevailing in infected rice fields in China. Leaf spot symptoms were observed in rice fields that were similar to those observed in other studies, i.e., brown-to-black fusiform-shaped lesions on leaves, branches, and pedicles [11]. In contrast to previous observations, the lesions observed in this study were surrounded by a yellow halo. Nigrospora species were initially considered as endophytic fungi [27,28] but were recently considered as the causal agent of leaf spot disease on rice plants [11]. Since then, many Nigrospora species have been identified as potential pathogens of new plant diseases on various host plants in different regions. This indicates that the Nigrospora species may threaten crop production. We should pay more attention to this species.
Recently, fungal identifications were based on morphology and molecular analysis, including the similarity of ITS or multi-genes [29]. However, the identification of the isolated isolates solely based on morphological characters does not seem to be reliable. Different fungi are separated using various multi-genes [2,29,30]. In Nigrospora species, the isolates were separated using the gene combination of ITS, TEF1-α, and TUB2 [3,4,11]. In this study, we also constructed ITS, TEF1-α, and TUB2 multigene-based identification and phylogeny analysis, which resulted in reliable species identification. A total of twenty-five isolates of Nigrospora species were classified into N. oryzae, N. sphaerica, N. chinensis, and a new species that we named N. anhuiensis. This shows that multigene phylogenetic analysis is a reliable and powerful method to differentiate morphologically difficult-to-identify fungal isolates. However, even this analysis method provides challenges for species identification, such as the high similarity of gene sequences, which makes the clear identification of some isolates more difficult. The identification method should, thus, receive further attention in the future.
Among the recovered species, we most often found N. oryzae, which is a well-known rice pathogen causing leaf spot disease [11]. However, we also isolated three additional species that were not known to be associated with leaf spot disease on rice: N. sphaerica, N. chinensis, and the newly identified species N. anhuiensis. We experimentally showed that all isolates were able to infect rice plants and cause spot lesions on the rice leaves and stalks. These results imply that rice leaf spot disease is not only caused by N. oryzae but also by N. sphaerica, N. chinensis, and N. anhuiensis. Furthermore, wild rice that produces wild rice shoots was widely cultivated around the rice fields in the Anhui province of China. The leaf spot disease caused by N. oryzae also occurred on wild rice leaves and grass, which may be another threat to rice disease control in Anhui province. These discoveries not only enlarge the known diversity of Nigrospora species in the Anhui province but also provide new clues to improve rice field management.
For new fungal species characterizations, combined morphological and molecular analyses are generally performed. However, there are few typical morphological characteristics for all the Nigrospora species. In our study, we discovered a novel isolate, QY-2, that has a smaller minimum conidia size, while the conidia size of N. oryzae isolates is bigger [11,17]. The conidia size is a special feature for fungal species identification. However, there are a few differences between the Nigrospora isolates, and the conidia size of the two N. oryzae isolates SS2-1A and SS-2-JB-2A are also different. Therefore, the morphological features are not reliable. The phylogenetic tree showed that QY-2 was classified into a single clade, suggesting it should belong to a new species named N. anhuiensis QY-2. The clade of QY-2 is closely related to N. oryzae, and the disease symptoms caused by the isolate QY-2 are also similar to those of N. oryzae following Koch’s postulates. Furthermore, our analysis showed that the isolate QY-2 is more sensitive to most tested fungicides than N. oryzae SS2-1A, suggesting that it can be easily controlled by general fungicides in the rice field. Therefore, it will likely not cause unpredictable yield loss when rice field management is carried out promptly.

New Species Description

Nigrospora anhuiensis Y. Liu & F. He, sp. nov.—Fungal Names|FN571838; Figure 2 and Figure 3. Named after the Anhui province of China, where this species was first collected. The isolate infects rice leaves and straw, causing leaf spot symptoms with a yellow halo. Hyphae hyaline, branched, septate. Conidia solitary, subglobose, black, shiny, smooth, aseptate, ellipsoidal, 11.715 − 13.998 × 9.62 − 11.418 μm (av. = 13.136 ± 0.815 × 10.574 ± 0.644). Colonies grow quickly on PDA and are initially white, followed by dark green with aging, reaching 6 cm in 2 d at 25 °C. On the carrot medium, the conidia will be produced in a week. Isolate of Nigrospora anhuiensis are classified in a distinct clade on a phylogenetic tree with a high support value (Figure 2).

5. Conclusions

In this study, we investigated the occurrence of the rice black leaf spot disease in the Anhui province of China and found that not only N. oryzae but also N. sphaerica, N. chinensis, or N. anhuiensis can cause rice black leaf spot disease. N. anhuiensis is a novel Nigrospora species based on a multi-locus phylogenetic tree and morphological analysis. The study provides new knowledge about rice leaf spot disease and suggests potential chemical agents to control rice black leaf spot disease in the field.

Author Contributions

Conceptualization, F.H. and G.Z.; methodology, F.H., Y.L., and J.A.; software, F.H. and Y.L.; validation, Y.L. and J.A.; formal analysis, Y.L.; investigation, Y.L., J.A., Y.S. (Yang Shen), Y.S. (Yang Sun), W.S., B.Z., X.T. and G.Z.; resources, F.H.; data curation, F.H.; writing—original draft preparation, F.H. and A.S.; writing—review and editing, F.H., A.S., G.Z., J.X. and J.S.; visualization, F.H. and Y.L.; supervision, F.H. and G.Z.; project administration, F.H.; funding acquisition, F.H. and G.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Major Science and Technology Projects in Anhui Province (No. 2020003a06020009) and the Anhui Provincial Natural Science Foundation (2008085MC77).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the data are covered in the paper and graphs.

Acknowledgments

The investigations were carried out in many cities, and we appreciated the manager of the rice field in each city.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Leaf spot symptoms caused by Nigrospora species in the field. (A) Grass leaf spot disease in the rice field. (B) Rice leaf spot symptoms. (C) Wild rice leaf spot symptoms.
Figure 1. Leaf spot symptoms caused by Nigrospora species in the field. (A) Grass leaf spot disease in the rice field. (B) Rice leaf spot symptoms. (C) Wild rice leaf spot symptoms.
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Figure 2. Phylogenetic tree based on the combined sequences of ITS, TEF1a, and TUB2. The ITS, TEF1-α, and TUB2 sequences were obtained from the NCBI database or by sequencing. The combined sequences were aligned using ClustalW2. The phylogenetic tree was generated through the neighbor-joining method with 1000 bootstrap replicates using MEGA 7.0. The isolates of this study are marked on the tree (black oval, N. oryzae isolates; red triangle, N. anhuiensis isolate; green triangle, N. sphaerica isolate; blue triangle, N. chinensis isolate). Size marker shows the evolutionary distances.
Figure 2. Phylogenetic tree based on the combined sequences of ITS, TEF1a, and TUB2. The ITS, TEF1-α, and TUB2 sequences were obtained from the NCBI database or by sequencing. The combined sequences were aligned using ClustalW2. The phylogenetic tree was generated through the neighbor-joining method with 1000 bootstrap replicates using MEGA 7.0. The isolates of this study are marked on the tree (black oval, N. oryzae isolates; red triangle, N. anhuiensis isolate; green triangle, N. sphaerica isolate; blue triangle, N. chinensis isolate). Size marker shows the evolutionary distances.
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Figure 3. Morphological features of five Nigrospora isolates. (A) The colony features of five typical isolates. The isolates were cultured on PDA medium, and pictures were taken at 2 days (2D) and 4 days (4D) post-inoculation. (B) Mycelial features. The isolates were cultured on glass slides for 24 h. Pictures were taken using a microscopy imaging system (Leica, Wetzlar, Germany). (C) Conidia features. The conidia were induced on carrot medium for one week and imaged using a microscopy imaging system (Leica, Wetzlar, Germany). The scale bar is shown in the photos.
Figure 3. Morphological features of five Nigrospora isolates. (A) The colony features of five typical isolates. The isolates were cultured on PDA medium, and pictures were taken at 2 days (2D) and 4 days (4D) post-inoculation. (B) Mycelial features. The isolates were cultured on glass slides for 24 h. Pictures were taken using a microscopy imaging system (Leica, Wetzlar, Germany). (C) Conidia features. The conidia were induced on carrot medium for one week and imaged using a microscopy imaging system (Leica, Wetzlar, Germany). The scale bar is shown in the photos.
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Figure 4. Comparison of conidia size between five Nigrospora isolates. The length (A) and the width (B) of the conidia were measured with the scale using a microscopy imaging system (Leica, Germany). The graph was drawn based on these data using Prism GraphPad 8.0 [26]. SS2-1A and SS-2-JB-2A: N. oryzae isolates; SS-2-JB-1B: N. sphaerica; QY: N. chinensis; QY-2: N. anhuiensis isolate. The shapes on the graph indicate the size of each conidia.
Figure 4. Comparison of conidia size between five Nigrospora isolates. The length (A) and the width (B) of the conidia were measured with the scale using a microscopy imaging system (Leica, Germany). The graph was drawn based on these data using Prism GraphPad 8.0 [26]. SS2-1A and SS-2-JB-2A: N. oryzae isolates; SS-2-JB-1B: N. sphaerica; QY: N. chinensis; QY-2: N. anhuiensis isolate. The shapes on the graph indicate the size of each conidia.
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Figure 5. Virulence of 19 isolates on the detached rice leaves. Isolates were grown on PDA for 2 days. Then, the fungal agar plugs from the colony rim were used to inoculate the 6-week-old rice leaves, and sterile agar plugs were used as the control (CK). Lesion lengths caused by each tested isolate were measured at 2 DPI. The lesion length data were analyzed, and the graph was drawn using Prism GraphPad 8.0 [26]. One-way ANOVA analysis was performed for lesion lengths using SPSS (SPSS version 25; IBM, Chicago, IL, USA) and QY-2 as the reference isolate. The red box indicates the five isolates from different groups that were also used for morphological analysis. a indicates p > 0.05, b indicates p < 0.05, and c means p < 0.01.
Figure 5. Virulence of 19 isolates on the detached rice leaves. Isolates were grown on PDA for 2 days. Then, the fungal agar plugs from the colony rim were used to inoculate the 6-week-old rice leaves, and sterile agar plugs were used as the control (CK). Lesion lengths caused by each tested isolate were measured at 2 DPI. The lesion length data were analyzed, and the graph was drawn using Prism GraphPad 8.0 [26]. One-way ANOVA analysis was performed for lesion lengths using SPSS (SPSS version 25; IBM, Chicago, IL, USA) and QY-2 as the reference isolate. The red box indicates the five isolates from different groups that were also used for morphological analysis. a indicates p > 0.05, b indicates p < 0.05, and c means p < 0.01.
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Figure 6. In vivo virulence of the novel N. anhuiensis QY-2 and other isolates. (A) The symptoms caused by five isolates on rice and wild rice. The fungal agar plugs were inoculated on the straw of the six-week-old rice leaves and three-month-old wild rice shoots, and sterile agar plugs were used as the control (CK). Lesion sizes were measured, and pictures were taken at 7 days post-inoculation. (B) The difference in lesion sizes between different isolates. The lesion length was analyzed and imaged using Prism GraphPad 8.0 [26]. QY-2 is the reference isolate. (C) Lesion length caused by N. oryzae isolate SS-2-B-2A. The fungal agar plugs were used to inoculate the straw of three-month-old wild rice shoots, and sterile agar plugs were used as the control. a indicates p > 0.05, b indicates p < 0.01.
Figure 6. In vivo virulence of the novel N. anhuiensis QY-2 and other isolates. (A) The symptoms caused by five isolates on rice and wild rice. The fungal agar plugs were inoculated on the straw of the six-week-old rice leaves and three-month-old wild rice shoots, and sterile agar plugs were used as the control (CK). Lesion sizes were measured, and pictures were taken at 7 days post-inoculation. (B) The difference in lesion sizes between different isolates. The lesion length was analyzed and imaged using Prism GraphPad 8.0 [26]. QY-2 is the reference isolate. (C) Lesion length caused by N. oryzae isolate SS-2-B-2A. The fungal agar plugs were used to inoculate the straw of three-month-old wild rice shoots, and sterile agar plugs were used as the control. a indicates p > 0.05, b indicates p < 0.01.
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Figure 7. Effects of different fungicides on N. anhuiensis isolate QY-2. The N. oyrzae (as the control) isolate SS2-1A and N. anhuiensis isolate QY-2 were cultured on PDA, adding different fungicides (carbendazim, mancozeb, difenoconazole, chlorothalonil, thiophanate–methyl, flusilazole, prochloraz, and thifluzamide) for 2 days at 25 °C. The colony diameter was recorded, and the data were visualized using GraphPad Prism 8.0 software [26]. One-way ANOVA analysis was performed for each concentration of the fungicides using SPSS (SPSS version 25; IBM, Chicago, IL, USA), and SS2-1A was used as the reference isolate. * indicates p < 0.05 and ** indicates p < 0.01.
Figure 7. Effects of different fungicides on N. anhuiensis isolate QY-2. The N. oyrzae (as the control) isolate SS2-1A and N. anhuiensis isolate QY-2 were cultured on PDA, adding different fungicides (carbendazim, mancozeb, difenoconazole, chlorothalonil, thiophanate–methyl, flusilazole, prochloraz, and thifluzamide) for 2 days at 25 °C. The colony diameter was recorded, and the data were visualized using GraphPad Prism 8.0 software [26]. One-way ANOVA analysis was performed for each concentration of the fungicides using SPSS (SPSS version 25; IBM, Chicago, IL, USA), and SS2-1A was used as the reference isolate. * indicates p < 0.05 and ** indicates p < 0.01.
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Table 1. Accession numbers of genes that used for phylogenetic analysis.
Table 1. Accession numbers of genes that used for phylogenetic analysis.
SpeciesIsolate IDGenBank Accession Numbers
ITSTUB2TEF1-α
N. oryzaeAQ-2OP677959PP103591PP103566
CZ-1AOP677960PP103592PP103567
CZ-1BOP677961PP103593PP103568
CZ-3AOP677963PP103594PP103569
CZ-3BOP677964PP103595PP103570
QY-1AOP677967PP103596PP103571
QY-1BOP677968PP103597PP103572
QY-3AOP677970PP103598PP103573
QYJ-4OP677971PP103599PP103574
SS1-2ZCAOP677972PP103600PP103575
SS1-2ZCBOP677973PP103601PP103576
SS1-WKL2OP677974PP112992PP103577
SS2-1AOP677975PP103602PP103578
SS2-1BOP677976PP103603PP103579
SS2-4OP677977PP103604PP103580
SS2-5AOP677978PP103605PP103581
SS-2-JB-2AOP677981PP103606PP103582
TL1-2AOP677982PP103607PP103583
TL1-2BOP677983PP103608PP103584
TL1-3OP677984PP103609PP103585
XC-1-LSD1OP677985PP103610PP103586
XC-LSD2OP677986PP103611PP103587
N. chinensisQYOP677966PP103612PP103588
N. sphaericaSS-2-JB-1BOP677980PP103613PP103589
Nigrospora sp.QY-2OP677969PP103614PP103590
Note: the rest of genes were downloaded from NCBI according to previous study [23]. The new isolate QY-2 was recorded as Nigrospora sp. in NCBI gene bank, but when the paper is published, the administrator will change the name.
Table 2. Origin of newly sampled Nigrospora isolates.
Table 2. Origin of newly sampled Nigrospora isolates.
IDDateCollection Area Latitude and LongitudeHost
SS1-2ZCA2021.09Yijiang District, Wuhu City *118°19′20.39″ E, 31°9′27.88″ NGrass
SS1-2ZCB2021.09Yijiang District, Wuhu City *118°19′20.39″ E, 31°9′27.88″ NGrass
SS1-WKL22021.06Yijiang District, Wuhu City *118°19′20.39″ E, 31°9′27.88″ NRice
SS2-1A2021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NRice
SS2-1B2021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NRice
SS2-42021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NRice
SS2-5A2021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NRice
SS-2-JB-1B2021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NWild rice
SS-2-JB-2A2021.09Yijiang District, Wuhu City *118°20′8.65″ E, 31°6′51.92″ NWild rice
TL1-2A2021.10Jiao District, Tongling City *117°37′29.60″ E, 30°48′49.95″ NRice
TL1-2B2021.10Jiao District, Tongling City *117°37′29.60″ E, 30°48′49.95″ NRice
TL1-32021.10Jiao District, Tongling City *117°37′29.60″ E, 30°48′49.95″ NRice
CZ-1A2021.10Guichi District, Chizhou City *117°26′10.27″ E, 30°38′51.64″ NRice
CZ-1B2021.10Guichi District, Chizhou City *117°26′10.27″ E, 30°38′51.64″ NRice
CZ-3A2021.10Guichi District, Chizhou City *117°26′10.27″ E, 30°38′51.64″ NRice
CZ-3B2021.10Guichi District, Chizhou City *117°26′10.27″ E, 30°38′51.64″ NRice
QY-1A2021.10Qingyang County, Chizhou City *117°55′15.66″ E, 30°34′58.69″ NRice
QY-1B2021.10Qingyang County, Chizhou City *117°55′15.66″ E, 30°34′58.69″ NRice
QY-3A2021.10Qingyang County, Chizhou City *117°55′15.66″ E, 30°34′58.69″ NRice
QY-12021.07Qingyang County, Chizhou City *117°53′57.06″ E, 30°36′45.36″ NRice
QY-22021.07Qingyang County, Chizhou City *117°54′49.77″ E, 30°36′5.07″ NRice
XC-1-ZSD12021.06Xuanzhou District, Xuancheng City *118°36′24.74″ E, 30°52′33.83″ NRice
XC-ZSD22021.06Xuanzhou District, Xuancheng City *118°36′24.74″ E, 30°52′33.83″ NRice
AQ-22021.06Huaining County, Anqing City *116°41′18.26″ E, 30°27′50.35″ NRice
QYJ-42021.06Huaining County, Anqing City *116°24′15.90″ E, 30°27′50.35″ NRice
* Anhui province of China.
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Liu, Y.; An, J.; Safdar, A.; Shen, Y.; Sun, Y.; Shu, W.; Tan, X.; Zhu, B.; Xiao, J.; Schirawski, J.; et al. Identification and Characterization of Nigrospora Species and a Novel Species, Nigrospora anhuiensis, Causing Black Leaf Spot on Rice and Wild Rice in the Anhui Province of China. J. Fungi 2024, 10, 156. https://doi.org/10.3390/jof10020156

AMA Style

Liu Y, An J, Safdar A, Shen Y, Sun Y, Shu W, Tan X, Zhu B, Xiao J, Schirawski J, et al. Identification and Characterization of Nigrospora Species and a Novel Species, Nigrospora anhuiensis, Causing Black Leaf Spot on Rice and Wild Rice in the Anhui Province of China. Journal of Fungi. 2024; 10(2):156. https://doi.org/10.3390/jof10020156

Chicago/Turabian Style

Liu, Yang, Jiahao An, Asma Safdar, Yang Shen, Yang Sun, Wenhui Shu, Xiaojuan Tan, Bo Zhu, Jiaxin Xiao, Jan Schirawski, and et al. 2024. "Identification and Characterization of Nigrospora Species and a Novel Species, Nigrospora anhuiensis, Causing Black Leaf Spot on Rice and Wild Rice in the Anhui Province of China" Journal of Fungi 10, no. 2: 156. https://doi.org/10.3390/jof10020156

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