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Article

Isolation, Identification and Characterization of Leptosphaerulina trifolii, the Causative Agent of Alfalfa Leptosphaerulina Leaf Spot in Inner Mongolia, China

by
Hongli Huo
1,2,3,
Jiuru Huangfu
2,
Peiling Song
2,
Dongmei Zhang
2,
Zhidan Shi
2,4,
Lili Zhao
4,5,
Ziqin Li
2,* and
Hongyou Zhou
1,3,*
1
College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010019, China
2
Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot 010031, China
3
Key Laboratory of Biopesticide Creation and Resource Utilization in Inner Mongolia Autonomous Region, Hohhot 010020, China
4
School of Life Sciences, Inner Mongolia University, Hohhot 010020, China
5
Tenihe Farm Co., Ltd., Hulunbuir 021024, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1156; https://doi.org/10.3390/agronomy14061156
Submission received: 23 April 2024 / Revised: 19 May 2024 / Accepted: 27 May 2024 / Published: 28 May 2024
(This article belongs to the Special Issue Diseases of Herbaceous Plants)
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Abstract

:
Leptosphaerulina leaf spot, caused by Leptosphaerulina trifolii, is a major disease of alfalfa (Medicago sativa), leading to noticeable losses. From 2022 to 2023, we collected samples of alfalfa with symptoms of the disease from different locations in Inner Mongolia, China. Nine fungal isolates recovered from these samples were identified through morphological traits and a maximum likelihood phylogeny based on concatenated partial sequences of ITS, 28S, and rpb2. A pathogenicity test on alfalfa confirmed the pathogenicity of the isolates on alfalfa. Analysis of physiological traits of L. trifolii revealed optimal mycelium growth at 20 °C and a pH range of 5 to 7, with soluble starch as the preferred carbon source and yeast extract as the optimal nitrogen source. The pathogen thrived in V8-juice agar and oat agar media. This study confirms L. trifolii as the causative agent of Leptosphaerulina leaf spot of alfalfa in Inner Mongolia and provides valuable insights into its optimal growth conditions. These findings enhance the understanding and management of this disease in alfalfa fields.

1. Introduction

Alfalfa (Medicago sativa L.), a perennial herb from the Papilionoideae family, is cultivated in more than 30 million hectares worldwide [1]. Recognized as the “king of forage crops”, it is distinguished by its valuable digestibility and high protein content, making it a highly nutritious feed choice for various livestock species [2]. Its rapid regrowth, resilience to environmental stresses such as drought and cold, and deep tap root system contribute to its ability to thrive in unmanaged environments [3]. Additionally, alfalfa contributes to nitrogen supplementation for both soil and plants, enhances soil organic matter, and improves soil structure [3,4]. Furthermore, this plant species has the potential for phytoremediation of soils contaminated with potentially toxic elements (PTEs) [5,6,7]. The increasing demand for meat, eggs, and milk in China has led to a significant expansion of alfalfa cultivation, especially in arid and semiarid regions of Northwest China in recent years.
Environmental factors, including biotic and abiotic stresses, pose major constraints on alfalfa production [8]. The impact of foliar diseases on alfalfa yield losses varied from 5.3 to 40%, with some instances of up to 52% reported [9,10,11,12]. Moreover, leaf spot injuries can lead to reduced photosynthesis and lower forage quality [13,14]. Research suggests that alfalfa foliar diseases on different parts of the plant are influenced by various environmental conditions [15]. Morsy et al. [16] discovered that salicylic acid, dipotassium phosphate (K2HPO4), and neem oil significantly reduced the severity of rust (Uromyces striatus J. Schrot., syn U. striatus var. medicaginis (Pass) Arth.) and downy mildew (Peronospora trifoliorum de Bary), respectively. Additionally, foliar fungicides have been found to increase yields under higher disease pressure [17].
Leptosphaerulina leaf spot on alfalfa can be caused by different Leptosphaerulina species, impacting both the alfalfa plant and the nutrient content of its leaves [18,19,20,21,22,23]. Liu et al. [21] first reported Leptosphaerulina leaf spot caused by L. trifolii on alfalfa in Heilongjiang Province, China, in 2019. Furthermore, this pathogen has been found to infect other plants, including taro (Colocasia esculenta), various annual Medicago species, subterranean clover (Trifolium subterraneum), and white clover (Trifolium repens), confirming cross-infectivity between alfalfa and white clover [9,24,25,26]. Despite these findings, there remains a lack of comprehensive research on the biology of L. trifolii makes it difficult to fully understand the pathosystem Leptosphaerulina/alfalfa.
In August 2022 and July 2023, a leaf spot disease was detected in alfalfa fields located in Hulunbuir and Chifeng City, Inner Mongolia. Infected leaves were collected and analyzed at a laboratory for identification of the causative pathogen. Investigative approaches encompassed morphological observation and molecular techniques, along with pathogenicity tests of the isolates, to ascertain and identify possible fungi associated with this type of disease symptom. Through this investigation, we characterized the biological traits of the pathogen and examined its mycelial growth in relation to factors such as temperature, pH levels, various carbon and nitrogen sources, and growth media. This enabled us to gain insight into the pathogen behavior during an outbreak in its natural environment, laying the groundwork for the development of strategies aimed at preventing and managing this disease.

2. Materials and Methods

2.1. Pathogen Isolation

Alfalfa leaves showing leaf spot symptom were collected from Hulunbuir and Chifeng Cities, Inner Mongolia of China in August 2022 and July 2023 (Table 1). Leaf samples were washed thoroughly under running tap water, and then randomly cut into multiple pieces with 2–3 mm in thickness at the boundary between healthy and diseased tissue. The sliced samples were sterilized using 75% ethyl alcohol for one minute and soaked in 5% sodium hypochlorite (NaClO) for two minutes [27], then immersed into sterile water for 30 s (thrice). Disinfected leaf pieces were plated on V8-juice agar medium supplemented with 40 µg/mL rifampin (dissolved in DMSO). The dishes were inverted and moved into a microbiological incubator at a steady temperature of 25 °C for 10 days without light.
Once colonies had grown around the tissue sections, the edges of the mycelium were cut and transferred to new Petri dishes containing V8-juice agar medium supplemented with 40 µg/mL rifampin. These plates were incubated for an additional 10 days. The isolates were then purified by using the hyphal tip method and transferring them onto 10% water agar medium, followed by V8-juice agar medium plates. The purified isolates were further processed and stored at 4 °C for further studies.

2.2. Morphological Identification

Nine isolates were chosen for morphological characterization and identification. These isolates were grown on a V8-juice agar medium containing 40 µg/mL rifampin, and incubated at 25 °C for 25 days until mature ascocarps were produced. Digital images of morphological features were captured using a Nikon upright fluorescence microscope eclipse Ni-U (Nikon Corporation, Tokyo, Japan) along with NIS-Elements Viewer 5.10.00 software (Nikon Corporation, Tokyo, Japan). The morphology, color, ascomata, asci, and ascospores were observed, measured, and photographed across 20 replicates. Isolates were identified based on morphological characterization of L. trifolii [21,25,28,29].

2.3. Molecular Identification

The internal transcribed spacer (ITS) nrDNA region, a fragment of the 28S rRNA gene (28S), and partial of the RNA polymerase II subunit 2 gene (rpb2) were amplified and sequenced using the primer pairs ITS1/ITS4 [30], LR0R [31] and LR5 [32], and RPB2-5F2 [33] and fRPB2-7cR [34], respectively (Supplementary Table S1). Total genomic DNA was extracted from fungal mycelia grown on V8-juice agar medium using Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology, Hangzhou, China). Amplification for each locus was conducted by following the polymerase chain reaction (PCR) protocols described by Valenzuela-Lopez et al. [35]. Each 25 µL PCR system contained 1 µL of DNA template (50 ng/µL), 1 µL of forward primer (10 nmol), 1 µL of reverse primer (10 nmol), 9.5 µL of ddH2O and 12.5 µL 2× EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China). PCR was performed using a VeritiTM 96-Well Thermal Cycler (Applied Biosystems, Singapore), ITS and 28S was amplified under the following reaction conditions: pre-denaturation at 94 °C for 30 s, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 1 min, and a final elongation at 72 °C for 3 min. For rpb2 amplification, a touchdown PCR program was applied following the description by Woudenberg et al. [36]. The PCR products were separated via electrophoresis on agarose gels running for 20 min in a TAE buffer. The PCR products were sequenced through a commercial sequencing service agent (BGI Tech Solutions, Beijing, China). Sequence primary identification and homology tests used the BLAST search tool on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi/ (accessed on 15 January 2024)) and then deposited in GenBank.
GenBank accession numbers of the isolates recovered from infected leaves of alfalfa during this study are listed in Table 2, and reference sequences of the Leptosphaerulina spp. described by Liang et al. [28] were selected for phylogenetic analyses (Supplementary Table S2). These sequences were retrieved, extracted, organized, and managed. Multiple sequence alignments were conducted using MAFFT V7 [37]. The aligned sequences of ITS, 28S, and rpb2 were input in order and then concatenated with the Concatenate Sequence tool after the Gblocks [38]. ModelFinder was employed to enhance the accuracy of phylogenetic estimates [39]. A phylogenetic tree was constructed using IQ-TREE (maximum likelihood) [40], with Phoma herbarium included as the outgroup, and branch support assessed using 1000 bootstrap replicates. These steps were carried out using the Phylosuite V1.2.3 software [41]. Subsequently, iTOL v6 webtool [42] was utilized to annotate phylogenetic tree based on the IQ-TREE results.

2.4. In Vitro Cultual Characteristics of Isolates

To understand the effect of temperature on the mycelium growth of isolates, the mycelial plugs were cultured on V8-juice agar medium at 5, 10, 15, 20, 25, 30, and 35 °C. In addition, the influence of pH on the growth of isolates was investigated. The V8-juice agar medium’s pH was modified using 1 N HCl and 1 N NaOH. Mycelial disks were then transferred onto the medium and cultivated at pH ranging from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Diverse nitrogen sources including glycine, KNO3, NH4Cl, (NH4)2C2O4, urea, (NH4)2SO4, L-methionine, yeast extract, beef extract, and tryptone were evaluated in a Czapek-Dox agar medium. As were different carbon sources including glucose, galactose, soluble starch, lactose, potassium acetate, glycerin, D-mannitol, maltodextrin, fructose, and sorbitol. The pH of different carbon and nitrogen source media is 6, and the dishes were inverted and moved into a microbiological incubator at a steady temperature of 20 °C. The test strains were placed in 10 different types of media: starch agar (SA), Czapek-Dox agar (CDA), malt extract agar (MEA), carrot agar (CA), potato sucrose agar (PSA), potato carrot agar (PCA), potato dextrose agar (PDA), oat agar (OA), V8-juice agar (V8J), and water agar (WA), and their formulation is listed in Supplementary Table S3. The colony diameters were measured using the intersecting method after 5, 10, 15, 20, and 25 days of incubation. The results from each treatment are presented as the mean of three replicates [43,44].

2.5. Pathogenicity Test

To examine the pathogenicity of the isolates in laboratory conditions, two experiments were conducted: in vitro leaf inoculation and potted plant inoculation [45]. Two-month-old “Hulunbuir variegated alfalfa” plants were prepared for in vitro leaf inoculation and potted plant inoculation, respectively. For in vitro leaf inoculation, the leaves with nearly identical sizes were removed from healthy plants and sterilized using 75% ethanol, then thoroughly washed with sterile water, followed by placing them on agar medium in Petri dishes. The fungal isolates were cultured on V8-juice agar medium for 25 days at 25 °C. A sterile cork-borer was used to place mycelial plugs (5 mm diameter) of isolates at the center of in vitro leaves, while control leaves were inoculated with sterile V8-juice agar medium discs. Each repetition involved 6 alfalfa leaves (three repetitions). Observed and photographed after 15 days.
A spore suspension was prepared with a concentration of approximately 1 × 106 spores/mL by scraping spores from the surface of V8-juice medium plates using sterilized cover glasses. The spore suspension, containing 0.2% Tween 80, was then sprayed onto the two-month-old “Hulunbuir variegated alfalfa” plants (5 mL per pot). Sterile water containing 0.2% Tween 80 served as control. Each repetition involved 20 alfalfa plants, with three repetitions for each treatment. The inoculated and control plants were maintained in a climate room at 25–28 °C and covered with plastic films to keep humidity. After two days, the plastic films were taken out and the plants were maintained in the climate room, then observed and photographed after 20 days. Infected leaves from two experiments were collected for isolation and identification of the inoculate fungal isolates to fulfill Koch’s postulates.

2.6. Statistical Analysis

The data underwent analysis of variance (ANOVA) based on a model for completely randomized design using the SPSS 26.0 software package (SPSS Inc., Chicago, IL, USA). Differences among means of treatments were evaluated using Duncan’s test at 0.05 probability level. Data are presented as the mean ± standard deviation.

3. Results

3.1. Disease Symptoms of Leptosphaerulina Leaf Spot and Fungal Isolation

Infected alfalfa samples were collected from Hulunbuir and Chifeng cities in Inner Mongolia, China, between 2022 and 2023 (Table 2). The symptoms observed on the leaves and stems of diseased alfalfa plants were tiny black pepper-like spots. Each lesion was usually surrounded by a chlorotic area. During the late stage of infection, the spots progressively spread throughout the leaves, leading to leaflet death (Figure 1). Nine Leptosphaerulina isolates (NM01–NM19) were recovered from the alfalfa plants showing leaf spot symptoms.

3.2. Morphological Identification of Isolates

The colony morphologies of all fungal isolates were similar. To observe asci and ascospores, ascocarps were transferred to a clean microscope slide. The ascocarps were spheroidal and subglobose, brown (Figure 2a), ranging in size from 87.60 to 232.38 × 71.21 to 229.41 µm (length × width), each containing several asci. The asci measured 58.56 to 76.96 × 9.66 to 20.70 µm, with each ascus containing eight hyaline immature or brown mature ascospores (Figure 2b). Ascospores were oval or obovate-elliptic, with two to four horizontal septa and zero to two vertical septa, ranging in size from 18.90 to 40.47 × 8.06 to 17.54 µm. Based on morphological characteristics, the nine isolates were tentatively identified as L. trifolii [21,25,28,29]. These isolates thrived well when grown on V8J medium at 25 °C, as depicted in Figure 2c,d. The colonies on V8J plates were round and flatted, with clear edges and radial growth, displaying a few white mycelia and numerous ascocarps scattered across or within the medium.

3.3. Molecular Identification

The ITS, 28S, and rpb2 DNA regions of the isolates were amplified through the PCR with ITS1 and ITS4, LR0R and LR5, and RPB2-5F2 and fRPB2-7cR primers and sequenced. All sequences of the nine isolates and alignment analysis based on the BLAST results indicated a 99.8 to 100% identity with reference isolates of L. trifolii. The phylogenetic tree created using concatenated sequences from ITS, 28S, and rpb2 regions, further confirmed the intimate relationship between our isolates and L. trifolii reference sequences. This distinction set them apart from other Leptosphaerulina species (refer to Figure 3), with the Phoma herbarium sequence positioned as the outgroup.

3.4. Cultural Characteristics of the Fungus

The colony diameters of L. trifolii isolates from alfalfa at different temperatures were measured at 5, 10, 15, and 20 days. Colonies grew between 5 °C and 30 °C, and the optimal growth temperature was 20 °C. Colony diameter reached 20.99 ± 0.41 mm and 43.49 ± 0.75 mm at 25 °C after 5 and 10 days, respectively. However, at 15 and 20 days, the largest colony diameters ranging from 65.92 ± 1.70 mm to 79.37 ± 0.99 mm were observed at 20 °C, which were significantly larger than the diameters of colonies of other temperatures (p < 0.05). Mycelium stopped growing at 35 °C (Figure 4a).
The mycelium diameters of L. trifolii isolated from alfalfa under the different pH treatments were measured at 5, 10, and 15 days. Colonies were able to grow between pH of 4 and 12 and preferred a slightly acidic to neutral environment. After 5 days, the maximum colony diameter was 29.83 ± 3.21 mm at a pH of 7, the difference was not significant with pH 5, 6, and 8 (p > 0.05), but was significantly different with rest pH treatments (p < 0.05). After 10 days, the maximum colony diameter was 52.92 ± 2.42 mm at a pH of 7, the difference was not significant with pH 5 and 6 (p > 0.05); however, it was significantly different with the other pH values (p < 0.05). After 15 days, the maximum colony diameter was 76.69 ± 1.78 mm at a pH of 6, the difference was not significant with pH 5 and 7 (p > 0.05), but was significantly different with the other pH values (p < 0.05). The isolates were not sensitive to alkalinity, as they can even grow under strong alkaline conditions. In acidic environments, the rate of mycelial growth decreased with a reduction in pH, and mycelium ceased to grow when pH dropped below 3 (Figure 4b).
As shown in Figure 4c, the rates of mycelial growth of the fungus with different carbon sources were obviously different. In the medium with soluble starch as a carbon source, the growth rate was the fastest: the colony diameter on 5, 10, 15, 20, and 25 days were 14.56 ± 0.04 mm, 28.71 ± 0.03 mm, 44.54 ± 0.24 mm, 58.01 ± 0.86 mm and 63.99 ± 1.02 mm, respectively, followed by medium with maltodextrin as the carbon sources. The rates of potassium acetate and fructose by the isolates were lower: the isolate stopped growing on the medium with potassium acetate as the carbon source because the colony diameters on 5, 10, 15, 20, and 25 days were 5 ± 0 mm (Figure 4c).
Using yeast extract as the nitrogen source resulted in the densest and fastest growth of mycelia, with average colony diameters of 18.49 ± 0.43 mm, 35.04 ± 0.22 mm, 52.51 ± 0.10 mm, 66.28 ± 0.47 mm, and 72.06 ± 1.41 mm after 5, 10, 15, 20, and 25 days, respectively. This was followed by medium with tryptone and beef extract as the nitrogen sources. They were significantly larger than the colonies under other nitrogen sources (Figure 4d). Therefore, soluble starch was the most appropriate carbon source for hyphal growth, and yeast extract was the best nitrogen source.
The isolate can grow on SA, CDA, MEA, CA, PSA, PCA, PDA, OA, V8J, and WA medium. The growth rate on V8J was the fastest as the colony diameters after 5, 10, and 15 days were 26.49 ± 0.62 mm, 49.66 ± 1.51 mm and 76.95 ± 1.35 mm, respectively, followed by OA with the colony diameters after 5, 10, and 15 days measured at 24.30 ± 0.28 mm, 47.19 ± 0.98 mm, and 75.12 ± 1.69 mm, respectively. This was significantly larger than the diameters obtained on other tested media (Figure 4e). The lowest colony growth was observed on CDA. Based on the results of the experiment, it appears that V8J and OA media may be the most suitable for promoting the growth of L. trifolii.

3.5. Pathogenicity Tests

In the in vitro leaf inoculation experiment, fifteen days after inoculation, some small black pepper spots were observed on the adaxial and abaxial side of alfalfa leaves inoculated with a spore suspension of isolate NM05 (Figure 5a,b), while the control remained symptomless (Figure 5c,d).
In the pathogenicity test on potted plants, it was observed that, 20 days after inoculation, older leaves showed numerous small black pepper-like spots with a chlorotic halo, which sometimes turned yellow and eventually fell off. Additionally, younger leaves displayed a few small black pepper-like spots. The disease progressed from the bottom to the top of the plants (Figure 5e). These lesions resembled those seen on field leaves. In contrast, control leaves remained free of symptoms (Figure 5f). Subsequently, the same fungus was successfully re-isolated from the inoculated symptomatic leaves, meeting the criteria of Koch’s postulates.

4. Discussion

Leptosphaerulina trifolii, a pathogen causing infections in alfalfa, was discovered in alfalfa fields near the sampling sites of Hulunbuir and Chifeng. This finding is supported by evidence from observations of alfalfa plant symptoms, morphological identification, multigene phylogenies, and pathogenicity tests. This marks the first recorded instance of L. trifolii infecting alfalfa in these regions, which had not been documented in China before 2015 [21]. Therefore, Inner Mongolia, China is a new territory of Leptosphaerulina leaf spot of alfalfa. This disease has also been linked to reduced alfalfa yields in Australia, North America, and other regions, leading to economic losses [14]. Disease symptoms of this disease are more severe on the lower old leaves and stems than the upper young leaves and stems under field conditions. One potential reason for this could be that the spores first infect the lower leaves and stems, then spread upwards to infect newer upper leaves and stems when conditions are conducive to re-infection.
rpb2 is considered to be the best loci recommended for rapidly identifying Leptosphaerulina species [28]. ITS and 28S are also used to detect L. trifolii [21,25,46]. In this research, the molecular identification of L. trifolii was firstly studied using the concatenated ITS, 28S, and rpb2 sequences, which improved the identification accuracy. L. trifolii can grow at a temperature ranging from 5 to 35 °C during in vitro cultivation. Olanya et al. [19] found L. trifolii had optimum growth rate at 20 °C; it is confirmed with our results. Other characteristics of L. trifolii have not previously been documented, such as pH, carbon and nitrogen sources, and culture conditions. The current study investigated these traits of L. trifolii for the first time.
Climate change can expand the geographic distribution of plant pathogen species or their vectors, which may lead to disease epidemics in new areas [47]. In addition, seeds, hay, and residues from infected alfalfa plants, insects, and farm machinery used in infected fields all promote the spread of plant diseases [48]. Fungicides continue to be a crucial tool in combating plant diseases. Ahonsi et al. [49] reported that cyproconazole, flutriafol, tebuconazole, and prothioconazole showed significant reductions in disease severity caused by L. chartarum. Lowe et al. [50] discovered that iprodione and propineb demonstrate effective control against L. trifolii in field conditions. Some advanced breeding lines and new varieties of annual Medicago species can be good resistance to L. trifolii in the field [51]. Alfalfa breeding for disease resistance, integrated farming practices, and new effective fungicides for Leptosphaerulina leaf spot will be a direction for the prevention and management of this disease.

5. Conclusions

The fungal species reported in this study was isolated from diseased alfalfa in China grasslands. The biological and molecular evidence, and pathogenicity tests confirmed that L. trifolii is one of the causes of alfalfa leaf spot disease in Inner Mongolia, China. The physiological traits reveal the appropriate conditions for fungal infection; this will enhance our knowledge of the pathogen. This fungus can grow well on V8J and OA medium in vitro and prefers soluble starch and yeast extract as the carbon and nitrogen sources. However, more studies of this fungus are required to better understand the potential impact of the disease on the leaves and stems of alfalfa. This study offers technical support for our future efforts to delve deeper into the spread of this pathogen in Inner Mongolia, China. Finally, integrated management strategies are essential to help the alfalfa industry, to minimize the impact of Leptosphaerulina leaf spot on this herbage, and to ensure crop sustainability.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14061156/s1. Table S1. List of primers used in this study. Table S2. List of GeneBank accession number of Leptosphaerulina species used for phylogenetic studies. Table S3. List of culture media used in this study. References [30,34,52] are also cited in Supplementary Materials file.

Author Contributions

Funding acquisition, Z.L.; Investigation, J.H., P.S. and L.Z.; Methodology, D.Z.; Resources, Z.S.; Supervision, H.Z.; Writing—original draft, H.H.; Writing—review & editing, Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA26050101-01), and Youth Innovation Fund Project of Inner Mongolia Academy of Agriculture and Animal Husbandry Sciences (2024QNJJN04).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank Yu CHEN (University of Saskatchewan, Canada) for critically reviewing the manuscript. We also thank Inner Mongolia Agricultural University biopesticide creation and utilization team for support.

Conflicts of Interest

Author Lili Zhao was employed by the company Tenihe Farm Co. Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Field symptoms of Leptosphaerulina leaf spot on alfalfa: Symptoms of Leptosphaerulina leaf spot on (a) aboveground parts, (b) leaves and (c) stems.
Figure 1. Field symptoms of Leptosphaerulina leaf spot on alfalfa: Symptoms of Leptosphaerulina leaf spot on (a) aboveground parts, (b) leaves and (c) stems.
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Figure 2. Microscopic identification of isolates (NM01-NM19). (a) Ascocarps on diseased leaves; (b) ascospore; fifteen-day-old L. trifolii colonies incubated at 25 °C on V8J (c) upper and (d) bottom. Scale bars: (a) = 200 µm; (b) = 20 µm.
Figure 2. Microscopic identification of isolates (NM01-NM19). (a) Ascocarps on diseased leaves; (b) ascospore; fifteen-day-old L. trifolii colonies incubated at 25 °C on V8J (c) upper and (d) bottom. Scale bars: (a) = 200 µm; (b) = 20 µm.
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Figure 3. Phylogenetic tree inferred from concatenated ITS, 28S, and rpb2 sequences of our isolates (in green background) and reference sequences using the maximum-likelihood method. Bootstrap values (%) presented at the branches were calculated from 1000 replications. Phoma herbarium was used as an outgroup. The scale bar indicates a 1% sequence difference.
Figure 3. Phylogenetic tree inferred from concatenated ITS, 28S, and rpb2 sequences of our isolates (in green background) and reference sequences using the maximum-likelihood method. Bootstrap values (%) presented at the branches were calculated from 1000 replications. Phoma herbarium was used as an outgroup. The scale bar indicates a 1% sequence difference.
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Figure 4. Optimization of culture conditions for Leptosphaerulina trifolii NM05. (a,b) V8-juice agar was used as the test medium; (c,d) Czapek-Dox agar was used as a replacement medium. (a) Effect of temperature on mycelial growth; (b) effect of pH on mycelial growth; (c) effect of different carbon sources on mycelial growth; 1, glucose; 2, galactose; 3, soluble starch; 4, lactose; 5, potassium acetate; 6, glycerin; 7, D-mannitol; 8, maltodextrin; 9, fructose; 10, sorbitol; (d) effect of different nitrogen sources on mycelial growth; 1, glycine; 2, KNO3; 3, NH4Cl; 4, (NH4)2C2O4; 5, urea; 6, (NH4)2SO4; 7, L-methionine; 8, yeast extract; 9, beef extract; 10, tryptone; (e) effect of different culture media. Means were compared using the Duncan test, with different letters above each bar indicating significant differences at p ≤ 0.05. Data are presented as the mean ± standard deviation of three replicates.
Figure 4. Optimization of culture conditions for Leptosphaerulina trifolii NM05. (a,b) V8-juice agar was used as the test medium; (c,d) Czapek-Dox agar was used as a replacement medium. (a) Effect of temperature on mycelial growth; (b) effect of pH on mycelial growth; (c) effect of different carbon sources on mycelial growth; 1, glucose; 2, galactose; 3, soluble starch; 4, lactose; 5, potassium acetate; 6, glycerin; 7, D-mannitol; 8, maltodextrin; 9, fructose; 10, sorbitol; (d) effect of different nitrogen sources on mycelial growth; 1, glycine; 2, KNO3; 3, NH4Cl; 4, (NH4)2C2O4; 5, urea; 6, (NH4)2SO4; 7, L-methionine; 8, yeast extract; 9, beef extract; 10, tryptone; (e) effect of different culture media. Means were compared using the Duncan test, with different letters above each bar indicating significant differences at p ≤ 0.05. Data are presented as the mean ± standard deviation of three replicates.
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Figure 5. Leaf spot symptoms caused by Leptosphaerulina trifolii (isolate NM05) on alfalfa. (a–d) in vitro leaf inoculation experiment and (e,f) inoculation test on potted plants. (a) Adaxial side of inoculated leaves after 15 days; (b) abaxial side of inoculated leaves after 15 days; (c) adaxial side of control leaves after 15 days; (d) abaxial side of control leaves after 15 days; (e) alfalfa plants inoculated and control in pots at 20 days; (f) leaves inoculated and control in pots at 20 days.
Figure 5. Leaf spot symptoms caused by Leptosphaerulina trifolii (isolate NM05) on alfalfa. (a–d) in vitro leaf inoculation experiment and (e,f) inoculation test on potted plants. (a) Adaxial side of inoculated leaves after 15 days; (b) abaxial side of inoculated leaves after 15 days; (c) adaxial side of control leaves after 15 days; (d) abaxial side of control leaves after 15 days; (e) alfalfa plants inoculated and control in pots at 20 days; (f) leaves inoculated and control in pots at 20 days.
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Table 1. Geographic origin of the fungal isolates characterized from alfalfa in this study.
Table 1. Geographic origin of the fungal isolates characterized from alfalfa in this study.
Isolate CodeLocationGeographical CoordinatesYear
LongitudeLatitude
NM01Hulunbuir, Inner Mongolia120°29′31″49°33′4″2022
NM03Hulunbuir, Inner Mongolia120°29′31″49°33′4″2022
NM05Hulunbuir, Inner Mongolia119°59′56′′49°20′41′′2022
NM06Hulunbuir, Inner Mongolia119°59′56′′49°20′41′′2022
NM08Chifeng, Inner Mongolia120°28′51′′43°36′37′′2023
NM11Chifeng, Inner Mongolia120°28′51′′43°36′37′′2023
NM14Hulunbuir, Inner Mongolia120°24′48′′49°30′8′′2023
NM16Hulunbuir, Inner Mongolia120°24′48′′49°30′8′′2023
NM19Hulunbuir, Inner Mongolia119°59′56′′49°20′41′′2023
Table 2. GenBank accession numbers of the isolates (L. trifolii) in this research.
Table 2. GenBank accession numbers of the isolates (L. trifolii) in this research.
IsolateGenBank Accession Number
ITS28Srpb2
NM01PP397170PP396740PP460948
NM03PP397171PP396741PP460949
NM05PP397172PP396742PP460950
NM06PP397173PP396743PP460951
NM08PP397174PP396744PP460952
NM11PP397175PP396745PP460953
NM14PP397176PP396746PP460954
NM16PP397177PP396747PP460955
NM19PP397178PP396748PP460956
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Huo, H.; Huangfu, J.; Song, P.; Zhang, D.; Shi, Z.; Zhao, L.; Li, Z.; Zhou, H. Isolation, Identification and Characterization of Leptosphaerulina trifolii, the Causative Agent of Alfalfa Leptosphaerulina Leaf Spot in Inner Mongolia, China. Agronomy 2024, 14, 1156. https://doi.org/10.3390/agronomy14061156

AMA Style

Huo H, Huangfu J, Song P, Zhang D, Shi Z, Zhao L, Li Z, Zhou H. Isolation, Identification and Characterization of Leptosphaerulina trifolii, the Causative Agent of Alfalfa Leptosphaerulina Leaf Spot in Inner Mongolia, China. Agronomy. 2024; 14(6):1156. https://doi.org/10.3390/agronomy14061156

Chicago/Turabian Style

Huo, Hongli, Jiuru Huangfu, Peiling Song, Dongmei Zhang, Zhidan Shi, Lili Zhao, Ziqin Li, and Hongyou Zhou. 2024. "Isolation, Identification and Characterization of Leptosphaerulina trifolii, the Causative Agent of Alfalfa Leptosphaerulina Leaf Spot in Inner Mongolia, China" Agronomy 14, no. 6: 1156. https://doi.org/10.3390/agronomy14061156

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