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

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The identification of Diaporthe tulliensis, Corynespora cassiicola, and Paramythecium foliicola as the causal agents of mulberry leaf spot in Hechi adds an important layer to our understanding of how fungal diseases move through intensive agricultural landscapes. Diaporthe is a genus of filamentous fungi that has long been recognized as a pathogen of soybeans, sunflowers, maize, and a wide range of fruit trees, and its ability to colonize a woody perennial host such as mulberry illustrates how flexible the host range of this group can be. Corynespora cassiicola, meanwhile, is one of the most polyphagous fungal pathogens known to plant pathology, documented on hundreds of plant species across tropical and subtropical regions, where it causes target spot on cotton, cucumber, tomato, papaya, and rubber, among many other crops. That both of these species appear as new records on mulberry suggests that the expansion and intensification of mulberry cultivation in Hechi may have created new opportunities for pathogens already circulating in the surrounding agroecosystem to jump onto a previously unexploited host. Paramythecium foliicola, by contrast, had already been reported as a mulberry pathogen in China, so its detection here confirms that it is an established component of the local disease complex rather than a recent arrival.

The methodology underlying these identifications reflects the current standard in fungal diagnostics, which combines classical morphology with multilocus DNA sequence comparison. The internal transcribed spacer region of ribosomal DNA, amplified with the ITS1/ITS4 primer pair, remains the universal barcode for fungi because it is present in high copy number, flanked by conserved priming sites, and divergent enough to distinguish most species. However, for genera such as Diaporthe and Corynespora, where closely related species can share nearly identical ITS sequences, the addition of protein-coding loci such as beta-tubulin (TUB2) and actin (ACT) provides the resolving power needed to assign isolates to the correct species. This is particularly important in Diaporthe, a genus in which species boundaries have been substantially revised in recent years as molecular phylogenies have revealed cryptic diversity hidden within what were once considered broadly defined morphological species. The three-locus approach used in this study therefore represents a pragmatic balance between rigor and feasibility, and it aligns with the recommendations of the international fungal community for describing and identifying plant-pathogenic fungi.

The confirmation of pathogenicity through Koch’s postulates deserves particular attention, because this step is frequently abbreviated or omitted in surveys of plant disease. By re-isolating the fungi from artificially inoculated leaves and reproducing the original symptoms, the researchers established a causal rather than merely correlational link between each fungus and the disease. This matters practically as well as scientifically: control measures aimed at the wrong organism waste resources and can allow the true pathogen to continue spreading unchecked. The selection of three highly pathogenic strains as target pathogens for the resistance screening also strengthened the subsequent evaluation, since a variety that withstands multiple independently virulent isolates is more likely to carry durable, broad-spectrum resistance than one that merely escapes infection by a single weak strain.

The detached-leaf inoculation method used to assess the 28 mulberry varieties is a well-established technique in plant pathology that offers several advantages over field-based screening. It allows precise control of inoculum concentration, humidity, and temperature, reduces the spatial requirements of large germplasm trials, and minimizes the influence of environmental noise that can obscure genuine differences in resistance. Detached-leaf assays have been used successfully in screening programs for many woody and herbaceous crops, and their results generally correlate well with field performance, although the correlation is rarely perfect because mature plants deploy structural defenses, such as leaf cuticle thickness and canopy microclimate effects, that excised leaves cannot fully reproduce. For this reason, the resistant varieties identified here would ideally be validated under natural field conditions over multiple seasons before being recommended for large-scale deployment, but the laboratory screen provides an efficient first filter that can prioritize the most promising candidates for further testing.

The finding that seven varieties—Gui 12, Gui 5, Wuguoganda, Nongsang 14, Jialing 30, Zhongsang 9703, and Hubeizijing—showed relatively high resistance against all three pathogens is notable because resistance to one fungal species does not necessarily predict resistance to another. Different fungal pathogens employ different infection strategies, deploy different suites of cell-wall-degrading enzymes and effector proteins, and are consequently countered by different layers of host defense. A variety that resists a necrotrophic pathogen such as C. cassiicola, which kills host tissue and feeds on the dead cells, may not resist a hemibiotroph such as Diaporthe, which initially maintains living host cells before transitioning to necrotrophy. The fact that these seven varieties performed well across all three target strains suggests they may possess constitutive defenses, such as preformed antimicrobial compounds or robust epidermal barriers, that are effective against a broad range of fungal attack strategies. Such broad-spectrum resistance is especially valuable in breeding programs because it is less likely to be overcome by shifts in the pathogen population.

The SSR marker analysis adds a population-genetic dimension that strengthens the practical value of the resistance screening. Simple sequence repeats are short tandemly repeated DNA motifs that mutate at high rates, making them highly polymorphic and well suited to distinguishing closely related cultivars. The clustering of the 28 varieties into four genetic groups, with resistant accessions separating from susceptible ones, hints at the possibility that resistance alleles are concentrated within particular genetic lineages. If this pattern holds up under further scrutiny, it would mean that breeders could use SSR markers as proxies to predict resistance in untested material, dramatically accelerating the screening process. More broadly, the dendrogram provides a map of the genetic diversity present in the Hechi germplasm collection, which is essential information for planning crosses: parents that are genetically distant tend to produce progeny with greater allelic diversity, increasing the chances of combining resistance with the agronomic traits—leaf yield, nutritional quality, and silkworm palatability—that determine commercial success.

The economic context of this work is difficult to overstate. Hechi’s mulberry plantation area of roughly 64,420 hectares makes it the largest sericulture-producing city-level region in China, and sericulture itself is a labor-intensive industry that supports millions of rural households across southern China. Mulberry leaves are not merely a substrate for silkworms but the sole determinant of cocoon quality and quantity, since silkworm growth rate, silk protein synthesis, and disease susceptibility are all directly tied to leaf nutritional status. The documented reductions in total sugar, reducing sugar, starch, protein, and lipid content in diseased leaves translate directly into slower larval development, smaller cocoons, and lower silk yield. A foliar disease that reduces both leaf biomass and leaf quality therefore strikes the sericulture economy twice, and the cumulative losses across a growing season can be substantial even when individual lesions appear minor.

The reliance on chemical fungicides such as carbendazim, mancozeb, and propiconazole for leaf spot control illustrates a broader tension in modern agriculture. These compounds are effective in the short term, but their repeated use selects for resistant pathogen strains, disrupts beneficial microbial communities on the leaf surface, and leaves residues that can harm non-target organisms. In sericulture the stakes are particularly high because silkworms are extremely sensitive to pesticide exposure; residues on mulberry leaves can cause larval mortality or sublethal effects that compromise cocoon production even at concentrations far below those lethal to the pathogen. This vulnerability makes host-plant resistance uniquely attractive for mulberry systems: a resistant variety requires no external chemical input, poses no residue risk to the silkworms feeding on its leaves, and remains effective season after season without the escalating costs associated with fungicide resistance management.

The comparison with resistance screening efforts in other mulberry-growing regions is instructive. Previous work assessing 85 germplasm lines against powdery mildew, Paramyrothecium leaf spot, and Pseudocercospora leaf spot found only four highly resistant lines, underscoring how rare strong resistance can be within available germplasm. The identification of seven varieties with relatively high resistance against three fungal pathogens in Hechi is therefore a meaningful outcome, though the moderate proportion of resistant material also reflects the genetic narrowness that often characterizes cultivated mulberry, where a limited number of elite cultivars have been propagated widely across growing regions. Expanding the screening to wild and semi-domesticated Morus accessions could uncover additional resistance sources, and the SSR framework established in this study provides a ready tool for characterizing the genetic relationships of any new material added to the collection.

Looking forward, the identification of these three pathogens opens several avenues for research. The genomes of D. tulliensis, C. cassiicola, and P. foliicola could be sequenced to identify candidate effector proteins and virulence factors, which in turn could be used to probe the molecular basis of resistance in the resistant varieties. Understanding which host recognition events trigger effective defense in varieties such as Gui 12 and Jialing 30 would enable marker-assisted selection, allowing breeders to track resistance genes through crossing programs without waiting for laborious pathogen inoculation assays at every generation. The SSR markers used here could also be supplemented with genome-wide single nucleotide polymorphism markers to increase the resolution of genetic maps and to identify quantitative trait loci associated with resistance. Because mulberry is a perennial outcrossing species with a large genome and a long generation time, molecular tools that accelerate the breeding cycle carry disproportionate value compared with annual crops.

The regional specificity of the findings also warrants emphasis. Pathogen populations differ substantially across mulberry-growing areas, as demonstrated by reports of Neophloeospora maculans as the principal leaf spot agent in Brazil and Paramyrothecium roridum in India, alongside Chinese records of Fusarium solani, Phoma bellidis, and Lasiodiplodia species. This geographic variation means that resistance screening must be tailored to the local pathogen community: a variety resistant to the pathogens circulating in one province may be fully susceptible in another where different species dominate. The Hechi study exemplifies the locally grounded approach that effective disease management requires, and its results should be interpreted as specific to the pathogen strains and environmental conditions of this region rather than as universally applicable recommendations. At the same time, the identification of two globally new host-pathogen records contributes to the worldwide catalog of mulberry diseases and may alert researchers in other regions to watch for the same organisms as sericulture continues to expand and intensify.

The timing of the sample collection, spanning June through December 2025 across six high-incidence orchards, also provides a template for disease surveillance in perennial fruit and leaf crops. Sampling across multiple months and sites captures the temporal and spatial heterogeneity of pathogen populations, which is essential for identifying the full spectrum of causal agents rather than the single dominant species at one moment. As climate patterns shift and cultivation practices intensify, the composition of fungal communities attacking mulberry may change, making periodic re-surveys a necessary component of long-term disease management. The combination of pathogen identification, resistance screening, and genetic characterization presented in this study offers a coherent model for how regional research programs can address emerging disease problems in a systematic and reproducible way, and the resistant varieties it has identified represent a tangible resource for the sericulture industry of Guangxi and beyond.

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

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

Article References: Yan, S., Zhang, S., Liu, J., Huang, K., Wen, L., Pan, L., Huang, X., Wu, W., Qin, Y., & Li, X. (2026). Identification of pathogens causing mulberry leaf spot and resistance evaluation of mulberry germplasms in Hechi, China. Frontiers in Agronomy, 8, Article 1930317. https://doi.org/10.3389/fagro.2026.1930317

Image Credits: AI Generated

DOI: 10.3389/fagro.2026.1930317

Keywords: Identification, pathogens, causing, mulberry, leaf, spot, resistance, evaluation, germplasms, Hechi, China, scientific research