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Frontiers in Agronomy ·

Volume 8 - 2026 | https://doi.org/10.3389/fagro.2026.1930317

Identification of pathogens causing mulberry leaf spot and resistance evaluation of mulberry germplasms in Hechi, China

  • Siyuan Yan1,2
  • Shihao Zhang1,2
  • Jiping Liu3
  • Kangdong Huang4
  • Liuying Wen5
  • Lingling Pan2
  • Xiumei Huang2
  • Weiwei Wu2
  • Yue Qin1,2*corresponding author
  • Xiaodong Li1,2*corresponding author
  1. 1Guangxi Key Laboratory of Sericulture Ecology and Applied Intelligent Technology, Hechi University, Hechi, China
  2. 2School of Chemistry and Bioengineering, Hechi University, Hechi, China
  3. 3Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agriculture University, Guangzhou, China
  4. 4Hechi Sericulture Technology Extension Station, Hechi Silkworm Egg Production Center, Hechi, China
  5. 5Baise Sericulture Development Center, Baise, China
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Abstract

Mulberry leaf spot, caused by fungal pathogens, is an economically devastating disease that leads to severe yield losses and reduces the nutritional quality of foliage, ultimately impairing the growth and development of silkworms. However, the causal agents of this disease in Hechi remain largely unidentified, and the availability of resistant mulberry cultivars is equally unclear. In this study, pathogens were isolated from symptomatic mulberry samples, and isolated strains were identified based on morphological characterization and molecular analyses of the ITS, TUB2, and ACT sequences. Following Koch’s postulates, the virulent isolates were recognized. Accordingly, three highly pathogenic fungal strains were selected as target pathogens and used to evaluate the disease resistance of 28 mulberry varieties by artificial inoculation of detached leaves with these pathogens. A set of 20 simple sequence repeat (SSR) markers was used to analyze 28 mulberry varieties. Our findings reveal that the causal agents of mulberry leaf spot in Hechi are Diaporthe tulliensis (SY53), Corynespora cassiicola (SY123), and Paramythecium foliicola (SY151), with D. tulliensis and C. cassiicola being new records worldwide. Among the 28 varieties tested, Gui 12, Gui 5, Wuguoganda, Nongsang 14, Jialing 30, Zhongsang 9703, and Hubeizijing exhibited relatively high resistance to all three strains. Furthermore, a dendrogram based on the 20 SSR marker set grouped the 28 varieties into four clusters, which separated resistant accessions from susceptible ones. This study identified the pathogens responsible for mulberry leaf spot in Hechi and screened disease-resistant mulberry varieties, providing a solid basis for the discovery of resistance genes and molecular breeding of mulberry.

Keywords

1. Introduction

Mulberry (Morus spp.) is a perennial woody plant belonging to the family Moraceae and is distributed widely in Asia, Africa, Europe, and other regions, with considerable economic value (Luo et al., 2024). China has been cultivating mulberry trees for over 5,000 years and is currently the country with the most mulberry species in the world (Zhu et al., 2023). In recent years, with the westward migration of the sericulture industry in China, Hechi’s sericulture industry has continued to expand, with mulberry plantation acreage growing steadily. By 2025, the mulberry planting area in Hechi had reached approximately 64,420 hectares, making it the largest sericulture-producing city-level region in China (http://www.hechi.gov.cn/). However, with the continuous expansion of mulberry cultivation area and the prolongation of its continuous cropping years, the incidence and severity of mulberry leaf spot have increased year by year (Zhao et al., 2026). Mulberry is of prime importance to the sericulture industry, as it serves as the primary food source for silkworms (Shair et al., 2020). The quality of mulberry leaves is one of the basic prerequisites for sericulture and plays a pivotal role in successful silkworm cocoon production (Ahmed et al., 2020). Compared with those in healthy mulberry leaves, the contents of total sugar, reducing sugar, non-reducing sugar, starch, protein, ash, and lipid were significantly decreased (Ghosh et al., 2003). Consequently, it poses a considerable impact on the health of the silkworm, directly affecting the economic returns of sericulture production (Jiang et al., 2019).

The pathogenic composition of mulberry leaf spot is complex, and the pathogen populations exhibit significant differences in different regions (Gs et al., 2023; Qazi et al., 2025). Pappachan et al. (2019) identified the causal agent of mulberry leaf spot in India as Paramyrothecium roridum based on morphological and molecular characteristics. Costa et al. (2020) demonstrated that Neophloeospora maculans was the principal causal agent of mulberry leaf spot in Brazil through morphological and molecular characterization, and pathogenicity evaluation. In China, Fusarium solani, Phoma bellidis, Lasiodiplodia sp., and P. foliicola have all been verified as capable of infecting mulberry leaves and causing leaf spots (Liu, 2019). Zhang (2023) clarified that N. maculans and Gonatophragmium mori are the pathogens causing leaf spot of mulberry.

To prevent and control mulberry leaf spot, growers primarily rely on chemical fungicides (Li et al., 2024). Specifically, carbendazim, mancozeb, and propiconazole are among the main fungicides used (Gs et al., 2023). The excessive use of chemical fungicides may lead to environmental hazards, drug residues, resistance of pathogen populations, and damage to non-target organisms, such as silkworms (Dev Maji, 2011). Therefore, resistance sources/varieties are the ultimate options to manage mulberry leaf spot (Banerjee et al., 2014). Genetic resistance provides an ideal solution for mulberry leaf spot control, and compared with chemical management, it is more cost-effective and poses fewer risks to the environment and public health (Sharma et al., 2025). Maji et al. (2009) assessed 85 mulberry varieties resistance against powdery mildew, Paramyrothecium leaf spot, and Pseudocercospora leaf spot under field conditions. Out of 85 germplasm lines, 4 lines were found highly resistant (HR), 9 lines were resistance (R), 43 lines were moderately resistant (MR), and 29 lines were susceptible (S). Powdery mildew severity showed a significant positive correlation with Pseudocercospora leaf spot. Screened varieties can be utilized for further disease resistance breeding. However, it should be noted that disease resistance evaluation is influenced by multiple factors, such as planting environment, agronomic management measures and pathogen types (He et al., 2026). Therefore, screening of leaf spot-resistant mulberry varieties tailored to the local conditions of Hechi is essential.

In this study, mulberry leaf samples with leaf spot symptoms were collected from six sites in Hechi between June and December 2025. The pathogens causing mulberry leaf spot disease in Hechi were identified via morphological examinations, molecular analyses, and pathogenicity tests. On this basis, three highly pathogenic fungal strains were selected as target pathogens, and the disease resistance of 28 mulberry varieties against these strains was evaluated. In addition to this, informative simple sequence repeat (SSR) markers were used to study genetic relatedness among the mulberry genetic resources. This study identified the pathogens responsible for mulberry leaf spot in Hechi and screened disease-resistant mulberry varieties, providing a solid basis for discovery of disease-resistant genes and molecular breeding of mulberry.

2. Materials and methods

Sample collection and isolation of the fungus

From June to December 2025, fifty-five diseased leaf samples of mulberry were collected from six high-incidence mulberry orchards in Hechi City, Guangxi Province, China (Figure 1). Mulberry leaf samples were washed with sterile water, surface sterilized in 75% ethanol for 60 s, 5% commercial NaClO for 3 min, washed four times with sterile water, and dried in sterile filter paper. The leaf tissue of these lesions was cut into small pieces (5 × 5 mm), plated onto potato dextrose agar (PDA) amended with ampicillin (Amp), and incubated at 25 °C in the dark for 3 ~ 5 d. Mycelium arising from explants was subcultured onto PDA medium to obtain pure cultures. In cases where small colonies grew from these samples, mycelia were picked from the edge and transferred to new PDA plates, and stored at 4 °C on PDA.

Morphological identification of fungal isolates

Pure cultures of the pathogen were grown on PDA at 25 °C for 7 d, with five replications per isolate. Cultural characteristics like colony color, pigmentation, texture, and growth were noted. Conidial morphology was observed using light microscopy.

DNA extraction and polymerase chain reaction amplification

Following the manufacturer’s instructions, mycelia were collected from the margins of pure cultures of each isolate grown on PDA, and total genomic DNA was extracted using Biospin Fungal Genomics DNA isolation Kit (Bioer Technology Co., Ltd., Hangzhou, China). DNA quantity was assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA), adjusting to a final concentration of 20 ng/μL, and stored at -20 °C until use. ITS, TUB2, and ACT were amplified using the primer pairs ITS1/ITS4, T1/Bt2b, and ACT-512F/ACT-783R (Supplementary Table 1). PCR was performed in a 25 μL reaction mixture containing 12.5 μL of 2 × Taq PCR Master mix (Sangon Biotech Co., Ltd.), 1.0 μL of each primer (10 μM), 1 μL of template DNA, and 9.5 μL of ddH 2 O. The amplification parameters were set as follows: initial denaturation at 98 °C for 10 min, followed by 34 cycles of denaturation at 98 °C for 30 s, annealing at 58 °C for 40 s, and extension at 72 °C for 1 min, with a final extension stage at 72 °C for 10 min. The cloned PCR products were Sanger sequenced at Sangon Biological Technology Company Limited.

Phylogenetic analysis

Homologous sequences of isolated ITS, TUB2, and ACT were obtained by querying the nucleotide sequences against the NCBI database using the BLAST search program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A maximum likelihood tree was generated based on the concatenated sequences of ITS + TUB2 + ACT sequences using MEGA X with the Jones Taylor Thornton model and 1000 replicates for bootstrap analysis.

Pathogenicity test

Pathogenicity experiment of isolated strains was performed as described by Liu et al. (2022). Pathogenicity tests were performed on detached leaves of healthy young mulberry plants of the variety Gui 6, which is widely cultivated in Hechi, China (Zeng et al., 2024). Mycelial plugs (5 × 5 mm) were taken from 14-day-old PDA cultures using a cork borer, and excess agar was trimmed. The mycelial plugs were placed on the adaxial surface of healthy mulberry leaves, with three inoculation sites per leaf at appropriate intervals. Each isolated strain was inoculated onto at least 10 leaves. Sterile PDA plugs were used as the negative control. Inoculated leaves were covered with a transparent polyethylene bag to keep the leaf surface moist. The inoculated leaves were incubated in a greenhouse at 25 °C, and the light duration was set to 14 h per day. The experiment was repeated three times.

Evaluation of mulberry germplasm for leaf spot resistance

28 mulberry germplasm varieties, including sericulture, fruit, and ornamental types, were selected for resistance screening against leaf spot caused by fungal pathogens (Supplementary Table 2). Mulberry leaves were collected from Mulberry Germplasm Nursery of Hechi University and Hechi Silkworm Egg Production Center. Each isolated strain was inoculated onto at least 10 leaves, with sterile PDA plugs as the negative control. For each isolate, the total number of inoculations and symptomatic lesions were recorded at 7 days post-inoculation (dpi), and the lesion area was measured. Given that chlorotic lesions evolve into necrotic lesions, the measurement of necrotic area included chlorotic lesions (Gonçalves et al., 2023; Guerrero-Egido et al., 2024).

Molecular characterization and diversity analysis

20 informative SSR markers were chosen for molecular characterization (Mathithumilan et al., 2013; Parida et al., 2016; Pinto et al., 2018) (Supplementary Table 3). The SSR genotyping was carried out in 25 μL of PCR reaction containing approximately 50 ng of template DNA, 12.5 μL of 2 × Taq PCR Master mix, and 10 pM of SSR primers. The amplification parameters were set as follows: initial denaturation at 98 °C for 10 min, followed by 34 cycles of denaturation at 98 °C for 30 s, annealing at 50 ~ 56 °C for 40 s, and extension at 72 °C for 1 min, with a final extension stage at 72 °C for 10 min. The final PCR product was separated on a 3% agarose gel. SSR marker data were numerically coded as presence = 1 or absence = 0, and missing data were coded as -1. The polymorphism information content (PIC) value was calculated from the formula given below: PIC = 1 – ∑(Pi) 2, where Pi is the proportion of samples carrying the i th allele of a particular locus.

Statistical analysis

Quantification of necrotic areas was performed using the image analysis software ImageJ. Statistical differences were evaluated by one-way ANOVA followed by Duncan´s post-hoc test. Significance was set at P < 0.05. The mean ± standard deviation (SD) was calculated by SPSS. Kappa index was used to assess concordance in SPSS. Cluster analysis was performed using the Ward method based on the square Euclidean distance by SPSS. Statistical analysis of the results was performed using GraphPad Prism version 11.0.1 with two-way ANOVA and Šídák’s multiple-comparison test. For SSR markers, the data formats were converted using DataFormater software. Genetic diversity parameters, such as effective allele number (Ne), gene diversity (H), and Shannon’s information index (I), were calculated with POPGEN v1.32. The SSR dendrogram of the 28 mulberry varieties was constructed with NTSYS-pc v2.1. The phylogenetic tree, heat map, and flower plot were visualized by using Chiplot (Xie et al., 2023).

3. Results

Symptom of Mulberry leaf spot

In 2025, mulberry leaf spot disease was first observed in Hechi City in June. By September, the disease had become widespread across all mulberry plantations, although the incidence varied among locations. The highest plant infection rate exceeded 70%, and the leaf infection rate surpassed 15%. By December, approximately 90% of the leaves had developed visible symptoms.

At the initial stage of leaf spot, translucent punctate lesions emerge on mulberry leaves. With disease development, the lesions expand progressively into circular or irregular brown necrotic lesions, accompanied by yellowing and chlorosis in the foliar tissues surrounding lesion margins (Figure 2A). In severely infected foliage, individual lesions merge and develop into large coalesced necrotic regions (Figure 2B).

Fungal isolation and pathogencity test

A total of 152 isolates, spanning 24 genera, were obtained from 6 sampling points of Hechi. Based on ITS sequences similarity, Paramyrothecium sp. was the most common species, representing 21.05% of all isolates, followed by Fusarium sp., Colletotrichum sp., Diaporthe sp., and Corynespora sp., which accounted for 19.74%, 18.42%, 8.55%, and 6.58%, respectively (Supplementary Table 4).

Pathogenicity assays were performed on ‘Gui 6’ leaves for all isolates to screen for major pathogens. Among the fungal isolates recovered from diseased leaves, 32, 10, and 8 strains of Paramyrothecium sp., Diaporthe sp., and Corynespora sp. showed pathogenicity. For the higher frequency and stronger pathogenicity, Paramyrothecium sp., Diaporthe sp., and Corynespora sp. were identified as the predominant causal pathogen associated with mulberry leaf spot in Hechi. Based on the sampling time, pathogenic isolates of Diaporthe sp. were predominantly recovered in June and September, yielding 6 and 4 strains, respectively. Corynespora sp. was mainly obtained in September and December, with 6 and 2 isolates, respectively. Paramyrothecium sp. was detected across all three sampling periods, with strain counts of 1, 23, and 8, respectively. Among all the pathogenicity isolates, SY53, SY123, and SY151 were found to be highly pathogenic and caused a maximum percentage of leaf spot. To confirm the causal agent, the pathogen was reisolated from the symptomatic tissues, and the resulting isolates were morphologically indistinguishable from the original inoculated strain (Supplementary Figure 1).

We monitored the necrotic lesion areas on Gui 6 leaves caused by isolates SY53, SY123, and SY151 at 7 and 10 dpi. Among the three isolates, SY151 exhibited the strongest pathogenicity. At 7 dpi, the necrotic area induced by SY151 reached 5.05 cm 2, which was significantly larger than those caused by SY53 and SY123. The lesions induced by all three pathogenic fungi expanded progressively with prolonged infection time. By 10 dpi, the necrotic area caused by SY151 had increased to 7.21 cm 2, remaining significantly larger than that of SY53 (Figure 3; Supplementary Figure 2).

Morphological and cultural characterization

The morphological characteristics of the three isolates were as follows. Colonies of strain SY53 were initially milky white with dense, short-floccose aerial hyphae, gradually turning from pale yellow to grayish-brown with age (Figure 4A1). The hyphae were smooth-walled (Figure 4B1). The α-type conidia were hyaline, unicellular, fusiform, and contained two globules, whereas the β-type conidia were hyaline, unicellular, filiform, with a hooked apical end and devoid of guttules (Figure 4C1). Strain SY123 formed grayish-white colonies with dense, velvety aerial mycelium and a brownish-yellow reverse, which later developed brick-red pigment deposits, while the reverse color shifted progressively from brownish-yellow to reddish-brown and ultimately to dark brown (Figure 4A2). The hyphae were septate (Figure 4B2), and the conidia were clavate and septate (Figure 4C2). Colonies of strain SY151 were initially white and velvety, eventually producing dark green, paint-like conidial masses arranged in concentric rings on the surface (Figure 4A3). The hyphae were smooth-walled (Figure 4B3), and the conidia were unicellular, bacilliform, with both ends bluntly rounded (Figure 4C3).

Phylogenetic analysis

We analyzed the phylogenetic relationships of the isolated pathogenic fungi based on combined ITS, TUB2, and ACT gene sequences. Isolate SY53, SY123, and SY151 formed a close cluster with D. tulliensis, C. cassiicola, and P. foliicola, respectively. Based on the integrated analysis of morphological characteristics and the ITS - TUB2-ACT phylogenetic tree, isolates SY53, SY123, and SY151 were identified as D. tulliensis, C. cassiicola, and P. foliicola, respectively (Figure 5).

Resistance screening of mulberry varieties against fungal leaf spot

Strains SY53, SY123, and SY151 were inoculated onto the leaves of 28 mulberry varieties, respectively. Each isolated strain was inoculated onto at least 10 leaves. The experiment was repeated three times. The results showed that the three pathogenic fungi exhibited differential pathogenicity on the leaves of the 28 mulberry varieties (Supplementary Table 5). However, no correlation was observed among the resistances of the 28 mulberry varieties to the three fungal pathogens.

The SY53 strain showed differential pathogenicity on the leaves of 28 mulberry varieties. At 7 dpi, Gui 6, Baichangguo, and Jialing 40 exhibited the largest lesion areas (1.46, 1.14, and 0.90 cm 2, respectively), demonstrating high susceptibility (HS) to SY53. Six varieties, including Yousang, Gui 5, Gui 12, showed lesion areas of 0.39 ~ 0.70 cm 2, indicating moderate susceptibility (MS) to SY53. Nineteen varieties, such as Gui 62, Jialing 30, Hongxisijiguo, showed lesion areas of 0 ~ 0.11 cm 2, indicating HR to SY53. Notably, fourteen of these varieties, including Dabaizhenzhu, Guiyou 2172, and Nongsang 14, were completely immune to SY53 infection (Figure 6A; Supplementary Figure 3).

SY123 showed differential pathogenicity on 28 mulberry varieties. At 7 dpi, Guiyou 2172 and Zhongshen 1 exhibited the largest lesion areas (7.83 and 6.57 cm 2, respectively), demonstrating HS to SY123. Five varieties, such as Gui 62, Guiyou 93200, and Kangqing 283, showed lesion areas of 2.32 ~ 3.87 cm 2, indicating S to SY123. Lesion areas of less than 2 cm 2 were observed in twenty-one varieties, including Jiuqulongsang, Qiangsang 1, and Yuesang 1, indicating HR to strain SY123. Furthermore, Nongsang 14 and Zhongsang 9703 were found to be completely immune to SY123 infection (Figure 6B; Supplementary Figure 3).

No complete resistance to strain SY151 was observed in any of the 28 mulberry varieties tested. At 7 days post-inoculation, the largest lesion areas were recorded for Yunsang 5 (13.74 cm 2), Jiuqulongsang (12.78 cm 2), and Kangqing 283 (11.63 cm 2), indicating that these three varieties are HS to the SY151 strain. Five varieties, such as Jialing 40, Zhongshen 1, Teyou 2, showed lesion areas of 8.55 ~ 10.10 cm 2, indicating MS to SY151. Lesion areas ranging from 5.00 to 7.67 cm 2 were observed in 13 varieties, including Yousang, Qiangsang 1, and Yuesang 51, indicating MR to strain SY151. Seven varieties, including Wuguoganda, Nongsang 14, Jialing 30, showed lesion areas less than 3.22 cm 2, indicating HR to SY151 (Figure 6C; Supplementary Figure 3).

Based on the differential pathogenicity of strains SY53, SY123, and SY151, the 28 mulberry varieties tested could be divided into six groups. Group I, comprising Yuesang 11, Dabaizhenzhu, Gui 6, Baichangguo, Yunguo, Taiwan Changguo, Yu 711, Yuesang 51, and Qiangsang 1, was characterized by MR to SY151. Group II, comprising Hongxisijiguo and Guiyou 93200, was characterized by MR to SY151, S to SY123, and HR to SY53. Teyou 2, Qiangsang 5, Yousang, and Jialing 40 were assigned to Group III, which was characterized by MR/MS to SY151 and HR to SY123. Zhongshen 1, Gui 62, and Guiyou 2172 were assigned to Group IV, which was characterized by HS/S to SY123, MR/S to SY151, and HR to SY53. Gui 12, Gui 5, Wuguoganda, Nongsang 14, Jialing 30, Zhongsang 9703, and Hubeizijing were assigned to Group V, which was characterized by relatively high levels of resistance against SY53, SY123, and SY151. Yunsang 5, Jiuqulongsang, and Kangqing 283 were assigned to Group VI, which was characterized by HS to strain SY151 (Figure 6D).

Cluster analysis

A total of 107 alleles were detected from 20 SSR markers and the number of alleles in varieties varied widely among the SSR markers. The number of alleles per locus ranged from two to nine, with an average of 5.35. The Ne varied from 1.07 to 7.28, with an average of 3.99. Observed heterozygosity (Ho) ranged from 0.26 to 0.70, with a mean of 0.42. The Expected heterozygosity (He) differed among the markers, and it ranged from 0.30 to 0.74, with an average of 0.58. I ranged from 0.42 to 2.72. The PIC value ranged from 0.26 to 0.72, with an average of 0.50 (Supplementary Table 6).

The SSR dendrogram of the 28 varieties was divided into four major clusters with coefficients ranging from 0.25 to 0.69 (Figure 7). At a genetic similarity coefficient of 0.338, the 28 mulberry varieties were divided into four clusters. Cluster 1 comprised fourteen varieties, cluster 2 comprised eleven varieties, cluster 3 comprised two varieties, cluster 4 comprised one accession.

Cluster 1 contained three SY53-HS varieties, including Gui 6, Baichangguo, and Jialing 40, accounting for 100% of all SY53-HS varieties. This cluster also contained twelve varieties showing HR/MR to the SY151 strain, including Gui 12, Gui 5, and Nongsang 14, representing 85.71% of the total varieties in Cluster 1. Cluster 2 comprised three SY151 HS varieties, like Yunsang 5, Jiuqulongsang, Kangqing 283, representing 100% of all SY151-HS varieties. According to the preliminary classification, Cluster 1 predominantly contained five varieties from Group I (5/9) and six from Group V (6/7), whereas Cluster 2 predominantly contained three strains from Group III (3/4) and all three varieties from Group VI (3/3). Notably, all 3 strains of Group VI were clustered together into a single cluster at a genetic similarity coefficient of 0.426. Consistency between SSR clustering and SY151 resistance was assessed by Kappa index, showing moderate concordance (Kappa = 0.415, P < 0.05).

4. Discussion

First report of D. tulliensis, and C. cassiicola causing mulberry leaf spot worldwide

The phyllosphere of terrestrial plants host a vast diversity of microbes, many of which are potentially pathogenic at certain stages of association with the plants (Yan et al., 2019). Mulberry suffers from a range of fungal diseases that result in significant yield losses and diminished leaf nutritional quality, ultimately inhibiting the healthy growth and development of silkworms (Ali et al., 2012). Mulberry leaf spot, caused by one or multiple fungal pathogens, exhibits significant variation in pathogen species composition depending on geographical regions and environmental conditions (Gs et al., 2023). Therefore, identifying region-specific pathogen species is critical for developing targeted disease management strategies. In our study, based on pathogen isolation, morphological characterization, molecular phylogenetic analysis, and Koch’s postulates via re-inoculation, we identified D. tulliensis, C. cassiicola, and P. foliicola as the causal agents of mulberry leaf spot in Hechi.

Species of Diaporthe are important plant pathogens with a wide range of hosts worldwide, causing leaf spot, stem rot, and stem blight of crops, orchards, and forests (Dissanayake et al., 2024). Maqbool et al. (2023) confirmed the involvement of D. ukurunduensis in mulberry leaf spot, in India. Diaporthe tulliensis has been reported to cause leaf spot in bodhi tree (Li et al., 2022) and betel vine (Shabong and Kayang, 2023), and leaf blight in coffee (Gong et al., 2020) and teak (Sazzad et al., 2026). In our study, D. tulliensis was found to infect leaves of mulberry, which might pose a potential threat to the establishment of mulberry plantations. To our knowledge, this is the first report of D. tulliensis causing leaf spot of mulberry in China and globally.

Paramythecium sp. is a pathogenic fungus distributed in tropical and subtropical regions, capable of infecting 32 economic crop species across 18 families, including Moraceae, Poaceae, Fabaceae, and Solanaceae. Most of its hosts exhibited classic leaf spot symptoms, although symptom severity and lesion morphology varied among host species (Aumentado et al., 2024). Mulberry leaf spot caused by Paramyrothecium sp. has been reported in Jiangsu (Tian and Li, 1993) and Guangdong Provinces (Liu, 2019), China, as well as in India (Pappachan et al., 2019). In our study, strain SY151, which was isolated and identified as P. foliicola, displayed the strongest pathogenicity among the three isolates. To date, leaf spot disease caused by this pathogen has not been documented in Guangxi.

Corynespora is a genus of plant pathogenic fungi with a global distribution, causing symptoms on leaves, stems, roots, flowers, and fruits of more than 400 plant species in over 70 countries (N. Rondon and Lawrence, 2021). C. Cassiicola could cause leaf spot in cotton (Salunkhe et al., 2019), Pogostemon cablin (Liao et al., 2025), and Ageratina altissima (Park et al., 2026). In this study, we obtained C. cassiicola SY123, which exhibited pathogenicity on multiple mulberry varieties. Notably, this is the first report of C. cassiicola causing mulberry leaf spot in China and worldwide.

The fungal leaf spot disease, caused by many different fungi, often exhibits similar pattern (Qazi et al., 2025). Tissue isolation results from this study demonstrated a temporal shift in the predominant pathogens causing mulberry leaf spot disease. In June, the major pathogens were Diaporthe sp. and Paramyrothecium sp., in December, the disease was mainly associated with Corynespora sp. and Paramyrothecium sp, while in September, all three fungal pathogens were present. However, the detection efficiency of this method is relatively low, making it inadequate to fully reveal the seasonal dynamics of the pathogens (Chavhan et al., 2018). Subsequently, species-specific detection assays will be established to ascertain their geographical distribution and to evaluate their associations with environmental variables, which may serve as a theoretical foundation for early surveillance and risk forecasting.

The resistance mechanisms of mulberry against different pathogens might be distinct

The main advantages of a detached leaf screening assay are the ability to minimize or eliminate environmental or field-based source of variation, such as temperature, humidity, and unintended microorganisms. Moreover, results from the in vitro assay correlate well with and are comparable to those from greenhouse and field screening across many fungus-plant pathosystems (Cowley et al., 2012; Sakr, 2022). In our study, the 28 mulberry varieties evaluated originated from two geographically distant sites. Various factors, such as temperature, agronomic practices, and nutritional status, may affect sporangia germination and infection by the pathogen, thus influencing the outcomes of the resistance evaluation (Neufeld and Ojiambo, 2012). Accordingly, we opted for the detached leaf assay as a controlled and standardized alternative to facilitate a more reliable comparison of resistance among the varieties tested. As a result, seven varieties, including Gui 12, Gui 5, Wuguoganda, Nongsang 14, Jialing 30, Zhongsang 9703, and Hubeizijing, were identified as exhibiting relatively high resistance to all tested three strains. It should be noted that although the detached-leaf assay effectively minimized environmental variation and revealed relative resistance differences among varieties, the results may not fully represent whole-plant resistance under field conditions. Therefore, further validation through multi-year and multi-season field trials is warranted.

The causal pathogens of mulberry bacterial wilt comprise a complex diversity of bacterial species, including Pantoea ananatis, Enterobacter cloacae complex, Klebsiella pneumoniae species complex, K. oxytoca complex, and Ralstonia pseudosolanacearum (Yuan et al., 2024). Kangqing 283 is a mulberry variety with high resistance to bacterial wilt, bred by the Zhanjiang Sericultural Science Institute (Luo et al., 2012). However, in the present experiment, all three tested strains were able to cause disease on its leaves, with the plant exhibiting S and HS responses to SY123 and SY151, respectively. This may be attributed to the differential resistance mechanisms of mulberry against fungal and bacterial diseases. Similarly, no correlation was observed among the resistances of the 28 mulberry varieties to the three fungal pathogens, suggesting that plant resistance mechanisms against different fungal diseases are distinct.

The molecular mechanisms involved in plant resistance towards pathogens have been extensively characterized in model species (Hulin et al., 2021). In this model, plants have evolved to perceive invading microbes and have acquired a sophisticated two-layered immune system (Dou and Zhou, 2012; Yu et al., 2019). The first, known as pathogen/microbe-associated molecular pattern (PAMP/MAMP)-triggered immunity (PTI/MTI), involves the recognition of PAMPs/MAMPs using pattern recognition receptors (PRRs) (Huang et al., 2018). PTI/MTI is a ubiquitous but weak form of resistance that can temporarily prevent infestation by most pathogens (Yang et al., 2022a). For successful infection, microbes can disrupt PTI/MTI by delivering effectors, leading to effector-triggered susceptibility (Rebaque et al., 2021). Plants use resistance proteins to detect these effectors, thereby initiating effector-triggered immunity (ETI) (Zhang et al., 2019a). The recognition of particular effectors by resistance proteins can activate plant defense responses. One of these defense responses is the hypersensitive response, a localized cell death reaction that limits or halts pathogen/microbe growth (Tarallo et al., 2022).

Deb et al. (2019) suggested that XopQ secreted by Xanthomonas oryzae pv. oryzae interacts with several rice 14-3–3 proteins and that this interaction is important for modulating rice innate immunity. Yang et al. (2022b) indicated that the conserved SnRK1A-XB24 module positively contributes to plant immunity. The virulence effector SCRE1, secreted by Ustilaginoidea virens, outcompetes SnRK1A for XB24 binding to promote infection. Liu et al. (2024) demonstrated that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Thus, it is reasonable to hypothesize that the three tested fungal pathogens secrete distinct effectors, which are recognized by distinct receptors in mulberry, thereby activating immune responses via different signaling pathways. This may explain the absence of correlation in disease resistance among the 28 mulberry varieties against the three tested strain. However, this hypothesis needs to be verified through further research.

SSR markers could identify leaf spot-resistant mulberry varieties

The analysis of genetic relationships among resistant and susceptible varieties is essential for their effective utilization in plant resistance breeding (Verma et al., 2019). A variety of molecular marker technologies, such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), SSR and single nucleotide polymorphism (SNP), have been widely used in the analysis of genetic diversity and plant variety identification (Bai et al., 2023). SSRs are short stretches of DNA consist of only one, or a few, tandemly repeated nucleotides that distributed throughout the eukaryotic genome (Huang et al., 2022). SSR markers display locus specificity, are co-dominant and highly transferable to other related species, and hence are the most attractive choice of marker systems for resistance evaluation (Zhang et al., 2019b; Duo et al., 2023). Meng et al. (2025) screened 8 SSR markers in maize, and this core marker set effectively separated resistant germplasms from susceptible ones. In mulberry, 16 SSR markers were employed to analyze 77 varieties with differential resistance levels. The dendrogram grouped the 77 varieties into two main clusters, with the majority of resistant varieties grouping separately from the susceptible ones (Arunakumar et al., 2021). In this study, 20 SSR markers were used to analyze the resistance of 28 mulberry varieties to three pathogens. The SSR clustering results were consistent with the resistance phenotype of mulberry against SY151, suggesting that these SSR markers could identify leaf spot resistance in mulberry. However, inconsistencies were observed in a small number of varieties. For instance, MR varieties such as Gui 62 and Jialing 40 were also clustered into Cluster 1. This may be attributed to the polygenic nature of resistance, and the current marker set may not fully cover the minor-effect resistance loci (Zhang et al., 2020). In addition, no consistency was observed between the marker set and the resistance phenotypes of SY53 and SY123, indicating that mulberry may exhibit differential resistance responses to distinct pathogens.

5. Conclusions

In conclusion, this study identified D. tulliensis, C. cassiicola, and P. foliicola as the causative agents of mulberry leaf spot disease in Hechi, with the former two representing new pathogen records for this disease. Resistance evaluation revealed that seven mulberry varieties, including Gui 12, Gui 5, Wuguoganda, Nongsang 14, Jialing 30, Zhongsang 9703, and Hubeizijing, exhibited broad-spectrum resistance to all three tested strains. Furthermore, SSR-based clustering could distinguish resistant from susceptible varieties, suggesting that these markers hold promise for marker-assisted selection in mulberry breeding programs. These findings fill critical gaps in the pathogen profile and host resistance of mulberry leaf spot, providing a foundation for disease management and resistance breeding.

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was received for this work and/or its publication. The present study was supported by the 2023 Annual Operation Subsidy Project of the Guangxi Key Laboratory of Sericulture Ecology and Applied Intelligent Technology (23-026-08), the 2026 Industry‑Education Integration Professional Cluster Project of Guangxi Ordinary Undergraduate Universities (Modern Agriculture and Biotechnology Professional Cluster), and the Yizhou District Sericulture Industry Science and Technology Chain Strengthening Project (AA2512260018).

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

SY: Supervision, Data curation, Project administration, Formal analysis, Validation, Writing – original draft, Methodology, Software, Conceptualization, Visualization, Investigation, Writing – review & editing, Funding acquisition, Resources. SZ: Writing – review & editing, Supervision. JL: Writing – review & editing, Supervision, Validation. KH: Writing – review & editing, Resources. LW: Writing – review & editing, Data curation, Resources. LP: Writing – review & editing, Resources. XH: Writing – review & editing, Resources. WW: Writing – review & editing, Resources. YQ: Writing – review & editing, Funding acquisition. XL: Writing – review & editing, Funding acquisition, Project administration, Resources.

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Citation

Yan, S., et al. (2026). Identification of pathogens causing mulberry leaf spot and resistance evaluation of mulberry germplasms in Hechi, China. Frontiers in Agronomy, Volume 8. https://doi.org/
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Copyright © 2026 Yan, Zhang, Liu, Huang, Wen, Pan, Huang, Wu, Qin and Li.

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*Correspondence: Yue Qin; Xiaodong Li

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