The research, published in MycoKeys, isolated pathogenic fungi from diseased leaves collected across multiple sites in Enshi Prefecture at approximately 575 metres above sea level. A total of 83 samples with clear disease symptoms were obtained and transported to the laboratory, where tissue segments containing both necrotic and healthy tissue were excised, surface-sterilised in 75% ethanol and 2% sodium hypochlorite, and cultured on potato dextrose agar. From these, 116 pure cultures were preserved and deposited in the Yangtze University Fungal Herbarium in Jingzhou, Hubei. The team then set out to determine exactly what these organisms were using a modern integrative taxonomic framework.

Fungal taxonomy has evolved dramatically from its early reliance on conidial phenotypic characters alone. High morphological similarity amongst species makes accurate identification based solely on phenotype challenging, and DNA sequence-based phylogeny has become the core approach, using standardised genus-specific multi-locus barcodes. For Coniella, the researchers sequenced ITS, LSU, RPB2 and TEF1; for Colletotrichum, ITS, CHS, ACT, CAL and TUB2; and for Pestalotiopsis, ITS, TEF1 and TUB2. Genomic DNA was extracted from fresh mycelia using the CTAB method, PCR amplifications were performed in 25 microlitre reaction volumes, and products were bidirectionally Sanger-sequenced. Yet even concatenated tree topologies have inherent limitations and a non-negligible risk of false positives, so the team went further, combining phylogenetic networks, SNP-based PCA and gene flow detection with cross-validation of GCPSR, DBSR and CBSR species recognition criteria.

The phylogenetic results were striking. For Coniella, a dataset of 2793 aligned characters revealed that four strains formed a distinct monophyletic clade as a sister group to C. castanea, with highly supported Maximum Likelihood bootstrap and Bayesian posterior probability values of 100/1.0 and no internal reticulate branches in phylogenetic networks. For Colletotrichum, 2344 aligned characters showed that two strains formed a distinct monophyletic clade within the Colletotrichum gloeosporioides species complex as a sister to Col. fructicola and Col. mengyinense. For Pestalotiopsis, 1276 characters placed two strains in the Pestalotiopsis rosea species complex as a stable independent clade closely related to P. rhodomyrti. All analyses consistently resolved the three species as independent taxa with highly-supported evolutionary lineages, distinct genetic clustering and significant nucleotide divergence.

Species delimitation was carried out using a hierarchical decision framework. Genealogical Concordance Phylogenetic Species Recognition evaluated topological congruence across individual single-gene genealogies, with recombination events assessed via the Pairwise Homoplasy Index test. Concordance-Based Species Recognition used Poisson Tree Processes and Bayesian PTP analyses running for one million MCMC generations, while Distance-Based Species Recognition applied Automatic Barcode Gap Discovery and Assemble Species by Automatic Partitioning under the K2-P model. A species boundary was formally accepted if corroborated by at least three independent delimitation methods. Notably, single-locus distance-based methods showed limited resolution in some cases; ABGD for LSU merged the Coniella target isolates with close congeners, and ASAP analyses failed to resolve boundaries within the Pestalotiopsis complex, highlighting the varying evolutionary resolution of individual barcode markers and the high sequence conservation within these genera.

SNP-based principal component analysis provided fine-scale genetic differentiation. SNPs were extracted from the concatenated multi-locus alignments using SNP-sites, and PCA conducted in R consistently revealed significant genetic differentiation between the present strains and their closely-related species. All tested strains formed distinct, well-separated clusters in PCA plots, fully concordant with the multi-locus phylogenetic and species delimitation results. This convergence of evidence across morphology, phylogeny, coalescent algorithms and genomic SNP data unambiguously validated three novel species: Coniella ampelopsidis, Colletotrichum enshiense and Pestalotiopsis ampelopsidis.

Morphologically, each species carries diagnostic characters. Coniella ampelopsidis produces hyaline, leguminiform to oblong conidia averaging 17.28 × 2.32 micrometres with a remarkably high length/width ratio of 7.54, clearly distinguishing it from C. castanea and C. quercicola. Colletotrichum enshiense, placed within the C. gloeosporioides species complex, is readily distinguished by its remarkably small, compact, ellipsoidal to ovoid conidia averaging 5.15 × 3.04 micrometres, far smaller than the elongated cylindrical conidia exceeding 11 micrometres typical of Col. fructicola. Pestalotiopsis ampelopsidis produces fusiform conidia with 3–4 septa averaging 22.50 × 6.22 micrometres, with barrel-shaped, verrucose, uniformly brown median cells and 2–3 apical tubular appendages, a unique combination separating it from P. rhodomyrti, P. oryzae, P. trachycarpicola and P. guiyangensis.

Pathogenicity assays fulfilled Koch’s postulates for all three species, confirming them as causal agents of leaf disease. Plants were acclimatised in a greenhouse at 25 °C with a 12-hour photoperiod, and living leaves were inoculated with 7-day-old mycelial plugs under high humidity exceeding 85%. Coniella ampelopsidis exhibited the strongest virulence, causing irregular yellowish-brown lesions with an average diameter of 20.8 ± 0.32 millimetres that spread rapidly and resulted in leaf necrosis. Pestalotiopsis ampelopsidis and Colletotrichum enshiense induced distinct leaf spot symptoms with average lesion diameters of 9.8 ± 0.10 and 10.2 ± 0.17 millimetres respectively, with no significant difference in virulence between them. Lesion area differences amongst strains were compared using one-way ANOVA and Tukey’s HSD post hoc test, and the pathogenicity was confirmed by re-isolation and ITS sequencing.

The phylogenetic patterns help clarify the evolutionary origins of these foliar pathogens. The closest relatives of Coniella ampelopsidis are primary pathogens on Fagaceae hosts, and as Fagaceae species form the dominant forest canopy in the mountainous ecosystems of Enshi Prefecture, spatial overlap and shared microclimates between oak forests and vine tea plantations likely facilitated ecological contact. In phytopathogenic fungi, sympatric proximity frequently provides opportunities for host jumping or host shifts, driving adaptive divergence and speciation. Similarly, Colletotrichum enshiense and Pestalotiopsis ampelopsidis are sister to broad-host-range pathogens, suggesting that ecological spill-over and host adaptation may have driven their lineage separation. Notably, this is the first formal report of a Pestalotiopsis species infecting plants from the genus Ampelopsis, expanding the known host range of the genus.

From an epidemiological perspective, the plausible origin via cross-host transmission from surrounding forest vegetation indicates that integrated disease management of vine tea should extend beyond field sanitation to incorporate surveillance of adjacent wild vegetation. The study enriches the inventory of foliar fungal pathogens infecting Nekemias grossedentata, which previously documented only four species, and demonstrates the effectiveness of an integrative taxonomic framework in resolving cryptic fungal pathogens on economic crops. Three phylogenetically distant fungal pathogens from three distinct orders co-occurring on a single host is a remarkable finding, and the scientific foundation it provides for regional disease monitoring and integrated management of vine tea may prove critical as this valuable medicinal crop faces mounting pressure from fungal disease.

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Subject of Research: Taxonomy and pathogenicity of three novel foliar fungal pathogens on Nekemias grossedentata in China

Article Title: Integrative taxonomy reveals three novel pathogens belonging to Coniella , Colletotrichum and Pestalotiopsis on Nekemias grossedentata in China

Article References: Chen, S.-Q., Zhou, X.-T., Mou, X., Zhou, S.-X., Deng, Q., Deng, J.-X., Luo, H., Chen, Q.-H., Liu, H.-F., & Zhang, C.-Y. (2026). Integrative taxonomy reveals three novel pathogens belonging to Coniella, Colletotrichum and Pestalotiopsis on Nekemias grossedentata in China. MycoKeys, 140, 49-77. https://doi.org/10.3897/mycokeys.140.200783

Image Credits: AI Generated

DOI: 10.3897/mycokeys.140.200783

Keywords: mycology, plant pathology, fungi, integrative taxonomy, phylogenetics, agriculture, Integrative, taxonomy, reveals, three, novel, pathogens

The kefir grain itself is a remarkable biological structure. It harbors more than 50 distinct microbial species, dominated by lactic acid bacteria including Lactobacillus helveticus, Lactobacillus kefiranofaciens, and Lactococcus lactis, alongside acetic acid bacteria, Bifidobacterium species, and yeasts such as Kluyveromyces marxianus, Candida, and Kazachstania. A large-scale analysis of 64 kefir grains from 25 countries revealed a strikingly consistent pattern: each grain is dominated by representatives of L. helveticus, L. kefiranofaciens subsp. kefiranofaciens, or L. lactis subsp. lactis or cremoris. This bacterial and eukaryotic heterogeneity is not incidental; the relative abundances of bacteria and yeasts directly influence the production of bioactive compounds. In water kefir, for example, coculture between L. kefiranofaciens and Saccharomyces cerevisiae enhances the synthesis of kefiran, a polysaccharide with documented antimicrobial activity.

The review’s central thesis is that kefir’s antimicrobial power derives from synergy, not from any single molecule. During fermentation, the microbial community hydrolyzes milk macromolecules, transforming them into metabolites with functional activity. Cell envelope proteinases of Lactobacillus catalyze the first step of casein hydrolysis, lactic acid bacteria consume lactose and drive the pH steadily downward, and acid-induced cleavage of the protein matrix releases bioactive peptides. On average, approximately 700 metabolites have been identified after fermentation. The resulting cocktail includes organic acids, bacteriocins, bioactive peptides, and exopolysaccharides, which act not only as individual molecules but as a complex combination of metabolic products that modify the environment and disrupt pathogen cell membranes or promote pores into bacterial cell walls.

Bacteriocins are among the most extensively characterized weapons in kefir’s arsenal. These ribosomally synthesized bacterial peptides, produced mainly by lactic acid bacteria such as Lactobacillus plantarum and Lactococcus species, range from less than 5 kilodaltons to more than 30 kilodaltons in molecular weight. Many exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures. Studies have demonstrated their activity against Klebsiella pneumoniae, Clostridioides difficile, methicillin-resistant Staphylococcus aureus, L. monocytogenes, P. aeruginosa, Salmonella enteritidis, Escherichia coli, Bacillus cereus, Enterococcus faecalis, and Cronobacter sakazakii. Metagenomic analyses of kefir grain samples have identified genes associated with bacteriocin synthesis, including helveticin and zoocin, and a bacteriocin designated ST8KF has been characterized from a kefir isolate of L. plantarum.

The molecular logic of bacteriocin action exploits a fundamental asymmetry between attacker and target. Both Gram-negative and Gram-positive bacteria present negatively charged surfaces, owing to the anionic lipopolysaccharide-rich outer membrane of the former and the negatively charged teichoic acids decorating the peptidoglycan of the latter. Bacteriocins with positive charge and hydrophobic domains bind electrostatically to these surfaces and insert into the membrane, forming pores that cause rapid cell death. Nisin, produced by L. lactis, employs a dual mechanism: it binds lipid II, a key component of peptidoglycan biosynthesis, disrupting the cell wall while simultaneously preventing peptidoglycan synthesis. Lactocin 27, from L. helveticus, inhibits protein synthesis without affecting DNA or RNA synthesis or ATP levels, and lactococcin 972 blocks septum biosynthesis rather than forming pores in the cytoplasmic membrane.

Kefiran, the signature exopolysaccharide of kefir, is a water-soluble branched glucogalactan composed of equal proportions of D-glucose and D-galactose, mainly produced by L. kefiranofaciens. Agar diffusion methods reveal its activity against Candida albicans and multiple bacteria, with the highest potency observed against Streptococcus pyogenes, while bacterial viability assays confirm bactericidal effects on P. aeruginosa, E. coli, S. aureus, and S. typhimurium. Using supported bilayer lipid membrane models, researchers showed that kefiran concentrations between 11.4 and 22.7 micromoles per liter induce membrane defects through penetration and pore formation, acting like a detergent. The proposed mechanism involves hydrogen bonding between the carbohydrate and the phosphate head group of the phospholipid, following a carpet-like model of interaction. Kefiran also protects intestinal epithelial cells by preventing Bacillus cereus-induced necrosis, F-actin disorganization, and microvilli effacement in Caco-2 cells, and has demonstrated potential against S. aureus in wound infections in a rat model.

Antimicrobial peptides represent another rich vein. Typically ranging between 6 and 50 residues, these small peptides destroy a wide range of microorganisms. Purification of 46 peptides from a Turkish milk kefir cultured with bovine milk, using reversed-phase high-performance liquid chromatography, showed that 13 achieved 100 percent inhibition against E. coli, K. pneumoniae, and P. aeruginosa. In a Brazilian kefir cultured with sheep’s milk, researchers identified 1,942 peptides using MALDI-TOF, of which 13 exhibited antimicrobial activity against P. aeruginosa, B. cereus, and B. subtilis, while ultra-high-performance liquid chromatography coupled with mass spectrometry revealed 257 peptides, 230 unique to kefir grains, sixteen previously described as bioactive. Critically, antimicrobial peptides have a significantly lower incidence of resistance compared with conventional antibiotics because they employ a multi-target mechanism, primarily the physical disruption of the bacterial membrane, in contrast to the single-target mechanism of traditional antibiotics.

Organic acids complete the picture. Lactic acid, the predominant end-product of lactose metabolism, decreases the milk pH from approximately 6.7 to less than 4.6, causing casein coagulation and inhibiting pathogen growth. Cell-free kefir supernatants contained lactic acid concentrations ranging from 83.59 to 229.92 millimolar and showed antagonistic effects on Salmonella arizonae and S. typhimurium. The antimicrobial mechanism of the undissociated form is elegant: the lipid bilayer is permeable to lipophilic acetic and lactic acids, which diffuse into the relatively alkaline cytoplasm, dissociate, and produce protons and anions. When protons exceed the buffering capacity, they are transported out through membrane-bound proton pumps, depleting energy reserves. The resulting lowering of intracellular pH denatures proteins and destabilizes structural and functional components of the cell. Lactic acid also potentiates other antibacterial molecules by disrupting the outer membrane of Gram-negative bacteria, making it permeable to metabolites that are too lipophilic or too large to penetrate otherwise, and sensitizing Gram-negative bacteria to bacteriocins such as nisin.

The kefir microbiota also possesses genetic potential to produce nonribosomal peptides and polyketides, secondary metabolites synthesized outside the ribosome by nonribosomal peptide synthetases and polyketide synthases. Bioinformatic prediction of biosynthetic gene clusters in metagenomic analyses of two Mexican kefir grain samples identified plipastatin, fengycin, and bacillaene, and detected contigs involved in exporting polyketides in Texas kefir metagenomes. In a Bacillus velezensis strain isolated from milk kefir, 55 percent of the genome was predicted to encode nonribosomal peptides, 36 percent polyketides, and 9 percent hybrids, with expression of genes for iturin, bacilysin, and mycosubtilin corroborated by RT-PCR, although experimental validation of their functional roles remains necessary.

The authors conclude that kefir grains represent an important source of antimicrobial compounds with biotechnological potential, but they caution that substantial heterogeneity in kefir preparation, dosage, intervention duration, study populations, and outcome measures limits interpretation of current findings. Larger, well-designed clinical trials with standardized kefir interventions, detailed reporting of microbial composition and viable counts, consistent dosing, longer interventions, and double-blind designs with matched sensory controls are needed to better define kefir’s efficacy in specific populations and health conditions. Nevertheless, the convergence of evidence from in vitro assays, lipid membrane models, metagenomics, and animal studies suggests that this ancient fermented drink may hold practical answers to one of modern medicine’s most urgent questions: where to find new antimicrobial molecules that pathogens have never encountered before.

Subject of Research: Minireview Kefir as a source of antimicrobial compounds against bacterial pathogens

Article Title: Minireview Kefir as a source of antimicrobial compounds against bacterial pathogens

Article References: Tenorio-Salgado, S., Sánchez-Canché, A., & Perez-Rueda, E. (2026). Minireview Kefir as a source of antimicrobial compounds against bacterial pathogens. Frontiers in Drug Discovery, 6, Article 1856085. https://doi.org/10.3389/fddsv.2026.1856085

Image Credits: AI Generated

DOI: 10.3389/fddsv.2026.1856085

Keywords: Minireview, Kefir, source, antimicrobial, compounds, against, bacterial, pathogens, scientific research

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

The receptor biology underlying HLA-G function deserves closer consideration because it determines which immune cell populations are affected when HLA-G appears in infected tissue. LILRB1 and LILRB2 belong to a family of immunoglobulin-like transcripts characterized by cytoplasmic motifs that, upon ligand binding, recruit phosphatases capable of dampening activating signaling cascades. When these receptors are engaged on natural killer cells, cytotoxicity is suppressed; when engaged on dendritic cells and macrophages, antigen presentation and pro-inflammatory cytokine production are attenuated; and when engaged on T lymphocytes, proliferation and effector function are curtailed. Because these receptors are distributed across both innate and adaptive lineages, a single tolerogenic molecule can exert coordinated suppression across multiple arms of the immune response simultaneously. This breadth of action distinguishes HLA-G from more narrowly targeted evasion mechanisms, such as fungal capsules that resist phagocytosis or enzymes that degrade complement, and explains why its exploitation would be particularly advantageous for pathogens seeking long-term persistence.

The damage-response framework referenced in the review offers a useful lens for interpreting why HLA-G induction during fungal infection could produce divergent clinical outcomes. According to this framework, the same pathogen-mediated immune alteration can be beneficial, neutral, or harmful depending on the host’s baseline immune status. In an immunocompetent individual, moderate HLA-G induction might serve as a homeostatic brake that prevents collateral tissue damage during fungal clearance, allowing the infection to be resolved without excessive inflammation. In a severely immunocompromised host, however, the same HLA-G activity could tip the balance decisively toward pathogen persistence by suppressing the residual antifungal effector mechanisms that remain. This context-dependence has practical implications: therapeutic interventions aimed at blocking HLA-G signaling would need to be calibrated to the patient’s immune status, since removing a compensatory regulatory mechanism in a hyperinflammatory patient could exacerbate tissue injury even as it enhances fungal killing.

The IL-10–HLA-G feedback loop described in the review warrants additional attention because self-amplifying circuits of this kind can establish stable immunosuppressive microenvironments that are difficult to reverse. When IL-10 induces HLA-G expression, and HLA-G in turn promotes further IL-10 production, the resulting positive feedback can lock infected tissue into a tolerogenic state even after the initial inflammatory trigger has subsided. This dynamic has been documented in chronic viral infections, where sustained IL-10 and soluble HLA-G levels correlate with impaired pathogen-specific T-cell responses and difficulty achieving immune reactivation. If a similar dynamic operates during chronic fungal infections—such as those caused by Candida species colonizing mucosal surfaces or by dimorphic fungi establishing latent infection—the feedback loop could contribute to the persistence and recurrence that characterize these conditions. Disrupting this loop at either node, whether through IL-10 neutralization or HLA-G receptor blockade, represents a potential host-directed therapeutic strategy that would enhance endogenous antifungal immunity rather than targeting the fungus directly.

The role of chitin as a fungal-derived trigger for IL-10 production illustrates how specific structural components of the fungal cell wall can actively shape the immunological tone of the host response. Chitin is normally buried beneath outer layers of glucans and mannoproteins, but it becomes exposed during cell wall remodeling, budding, and damage caused by antifungal drugs. Once accessible, chitin engages pattern recognition receptors including the mannose receptor, NOD2, and Toll-like receptors, and this coordinated signaling preferentially drives IL-10 rather than pro-inflammatory cytokines. This is a striking example of fungal structural biology being repurposed for immune evasion: a molecule that serves an essential structural role in the cell wall simultaneously functions as an immunoregulatory trigger when exposed. The clinical implication is that antifungal treatment itself, by damaging cell walls and exposing chitin, might inadvertently enhance the very immunosuppressive pathways that facilitate persistence, suggesting that combination approaches pairing antifungals with immunoregulatory modulation could be more effective than either alone.

Transforming growth factor-β, the second cytokine highlighted in the review, operates through partially overlapping but mechanistically distinct pathways compared with IL-10. While IL-10 primarily suppresses antigen-presenting cell function and Th1 responses, TGF-β exerts broader effects on immune cell differentiation, promoting the generation of regulatory T cells and inhibiting the differentiation of pro-inflammatory Th17 cells that are important for mucosal antifungal defense. The convergence of IL-10 and TGF-β on HLA-G expression creates a scenario in which multiple regulatory cytokines induced during fungal infection could act synergistically to establish a tolerogenic microenvironment. This convergence is significant because it means that blocking any single cytokine pathway may be insufficient to prevent HLA-G induction; instead, combination strategies targeting multiple regulatory axes simultaneously may be required to fully restore protective antifungal immunity.

The concept of host-directed therapy, which the review identifies as a potential application of this research, represents a growing paradigm in infectious disease management. Rather than developing new antifungal compounds that target pathogen-specific molecules—a strategy constrained by the limited number of druggable fungal targets and the toxicity of many existing agents—host-directed approaches modulate the host environment to make it less permissive for pathogen survival. Blocking HLA-G signaling, neutralizing IL-10 or TGF-β, or disrupting the feedback loops that sustain immunosuppression could restore the efficacy of existing antifungal drugs by reactivating the host’s own effector mechanisms. This approach also has the advantage of being applicable across multiple fungal species, since the host pathways being targeted are shared rather than pathogen-specific.

The translational gap between the mechanistic framework proposed in this review and clinical application remains substantial. Direct evidence that HLA-G is upregulated during human fungal infections is currently limited, and the review’s argument is built largely on the convergence of well-established pathways from other infectious and inflammatory contexts. Demonstrating that HLA-G expression is measurably elevated in tissue biopsies or serum samples from patients with invasive candidiasis, aspergillosis, or other fungal diseases would be a critical first step. Subsequent studies would need to establish that this elevation correlates with clinical outcomes such as treatment failure, fungal persistence, or disease severity, and that interventions targeting the HLA-G pathway can improve outcomes in relevant models. The heterogeneity of fungal infections—with different species, anatomical sites, and host immune contexts potentially producing different HLA-G dynamics—means that the relationship will likely need to be characterized individually for each major fungal pathogen rather than assumed to be universal.

The soluble isoforms of HLA-G, particularly HLA-G5, deserve specific consideration in the context of fungal disease because they can circulate systemically and exert immunoregulatory effects at sites distant from their cellular source. If fungal infections induce soluble HLA-G release from monocytes or other cell types, this could contribute to the systemic immunosuppression observed in invasive fungal disease, potentially explaining why disseminated fungal infections are associated with broad defects in cell-mediated immunity that extend beyond the local site of infection. Measuring soluble HLA-G levels in the serum of patients with invasive mycoses could therefore serve both as a biomarker of disease severity and as a mechanistic indicator of the immunoregulatory processes driving pathogenesis.

The genetic variability of the HLA-G locus adds another dimension to this question. The HLA-G gene is polymorphic, and certain alleles are associated with higher or lower expression levels of both membrane-bound and soluble forms. If fungal pathogens do indeed exploit HLA-G-mediated tolerance, individuals carrying high-expressor alleles might be disproportionately susceptible to fungal persistence or chronic infection, while those with low-expressor variants might be relatively protected. Conversely, the same high-expressor alleles might protect against immunopathology in settings where fungal-induced inflammation is excessive. Understanding how HLA-G polymorphisms influence susceptibility to fungal disease could open new avenues for risk stratification and personalized management of patients at high risk for invasive mycoses, such as those undergoing hematopoietic stem cell transplantation or receiving prolonged immunosuppressive therapy.

The comparison between fungal and viral exploitation of HLA-G is instructive because it highlights both the plausibility of the hypothesis and the specific challenges that fungal infections present. Viruses, as intracellular pathogens, can directly manipulate host gene expression through viral proteins that interfere with transcriptional regulation, making it straightforward to envision how they might upregulate HLA-G. Fungi, as predominantly extracellular pathogens, lack this direct access to the host nucleus and must instead rely on indirect mechanisms—such as inducing host cytokines like IL-10 and TGF-β, or releasing cell wall components that trigger regulatory signaling—to create conditions permissive for HLA-G expression. This indirect route is less efficient but potentially more difficult to block, since it involves multiple host pathways rather than a single viral effector. It also means that the timing and magnitude of HLA-G induction during fungal infection would depend on the complex interplay between fungal burden, host cytokine production, and tissue microenvironmental factors, making the relationship more variable and context-dependent than in viral systems.

The broader significance of this hypothesis extends beyond fungal pathogenesis to the general question of how immune checkpoint pathways are co-opted across infectious diseases. If HLA-G serves as a convergent target for diverse pathogens—viruses, parasites, and potentially fungi—this suggests that it represents a fundamental vulnerability in host defense that pathogens have independently discovered through convergent evolution. Understanding the common principles governing this exploitation could inform therapeutic strategies that are broadly applicable across multiple infectious diseases, rather than being specific to a single pathogen. It also raises the possibility that other immune checkpoint molecules beyond HLA-G, such as programmed death ligand 1 or cytotoxic T lymphocyte-associated antigen 4, may similarly be exploited during fungal infection, creating a network of overlapping immunosuppressive mechanisms that collectively enable persistence.

Future research directions emerging from this framework include the systematic measurement of HLA-G expression in clinical specimens from patients with defined fungal infections, the use of in vitro models to test whether fungal stimuli directly or indirectly induce HLA-G in human monocytes and dendritic cells, and the development of animal models in which HLA-G signaling can be experimentally manipulated to determine its causal role in fungal persistence. Each of these approaches addresses a specific gap in the current evidence base, and together they would provide the foundation needed to translate the mechanistic hypothesis into clinically actionable insights. The intersection of fungal immunology and immune checkpoint biology remains largely uncharted territory, and the framework presented here offers a structured starting point for systematic investigation.

Subject of Research: Immunology

Article Title: Do fungal pathogens exploit HLA-G-mediated immune tolerance?

Article References: AlSaleh, A., & Farid, E. (2026). Do fungal pathogens exploit HLA-G-mediated immune tolerance?. Frontiers in Molecular Biosciences, 13, Article 1963030. https://doi.org/10.3389/fmolb.2026.1963030

Image Credits: AI Generated

DOI: 10.3389/fmolb.2026.1963030

Keywords: HLA-G, fungal infections, immune evasion, IL-10, TGF-beta, host-directed therapy, fungal, pathogens, exploit, HLA-G-mediated, immune, tolerance

The work, published in the journal Individual-based Ecology, set out to answer a deceptively simple question: do pigeons that move differently also carry different microbes? The feedback between host behavior and disease transmission is widely acknowledged in theory, but empirical studies demonstrating the strength and direction of the association between an individual’s pathogens, its microbiota composition and its movement remain rare. Pigeons, known reservoirs for agents such as Salmonella, Chlamydia, Cryptococcus and Escherichia, offered an ideal test case because they overlap constantly with people at urban roosts and with livestock at agricultural sites.

The team captured 308 pigeons between July 2019 and November 2021 at three dairy farms arranged along an urbanization gradient: Mevo Horon, an isolated rural settlement 13 kilometers northwest of Jerusalem; Ma’ale Hahamisha, a semi-rural kibbutz on an elevated ridge surrounded by several small settlements; and Mikve Israel, on the outskirts of the Tel Aviv metropolitan area, where 42 percent of the surrounding land is urban. Adults in good body condition above a 250-gram threshold were fitted with OrniTrack GPS transmitters in a backpack configuration, which sampled locations every ten minutes during daylight when batteries allowed. In total, 44 pigeons were tagged, and 32 accumulated 8,473 days of tracking data — an average of 265 days per individual, with some birds followed for more than two years.

Microbiome sampling relied on a combined swabbing of the choanal cleft and cloaca of every captured bird, preserving samples on paper cards for RNA extraction. From the tagged subset, 29 samples went through untargeted total RNA sequencing on a MiSeq machine — an approach the authors chose because it outperforms 16S amplicon sequencing and shotgun metagenomics by detecting viral and bacterial sequences outside the 16S locus, giving higher discriminatory power. Taxonomic profiling filtered results with strict cutoffs to limit false positives, and bacteria were analyzed primarily at the genus level, where classification accuracy is greatest. The researchers validated the sequencing results against classical colony-growth tests for Campylobacter and Salmonella, finding strong agreement between methods.

The movement data revealed a clear urban effect. Pigeons at the rural and semi-rural sites traveled roughly 2,100 to 2,500 meters per day on average, with maximum daily displacements exceeding 3,000 meters, while pigeons at the urban Mikve site covered only about 1,000 meters daily and displaced barely 1,300 meters from their roosts. The differences were statistically significant for both travel distance and displacement. The likely explanation is resource distribution: rural pigeons spent summer months foraging for grains at harvested fields far from their roosts, while urban birds foraged year-round on human waste and rarely visited distant fields. This pattern agrees with broader findings that urban animals move less thanks to stable, rich food sources.

All pigeons, regardless of site, roosted almost exclusively in buildings within human settlements and routinely visited their respective dairy farms and other anthropogenic food sources such as parks, poultry farms and commercial areas. Crucially, crossing among the three study sites was very rare, meaning each farm’s pigeon population operated largely as a closed local system — yet one that alternated constantly between urban roosts and livestock facilities, sometimes located within hundreds of meters of each other.

The microbiome results were equally striking. The top 20 most abundant bacterial genera across all sites included Escherichia, Campylobacter, Clostridium and Streptococcus — genera containing many species of disease-causing pathogens. At the species level, the researchers identified bacteria with documented illnesses in humans, cattle and poultry, confirming that the pigeons carry not just pathogen-relevant genera but pathogenic species themselves. Local studies have previously reported Salmonella and E. coli in Israeli pigeons, and the new sequencing data are consistent with that picture of substantial zoonotic potential.

Site-level differences in microbial diversity followed the urbanization gradient in an unexpected direction. Pigeons at the urban Mikve site carried significantly higher bacterial richness than those at the two rural sites, with around 160 genera per bird compared to roughly 70 at Mevo and Ma’ale Hahamisha. Fisher’s alpha diversity, a measure that accounts for evenness, showed the same trend, though it was not statistically significant across all indices. The authors caution that with only three sites, factors such as elevation, resource composition and local density cannot be ruled out, but the urbanization gradient is the obvious candidate explanation, and prior work has attributed increased microbial diversity in urban wildlife to parasite acquisition, natural infections and physiological stressors at urban habitats.

The study’s most novel finding concerns individual behavior. Among 19 pigeons with both high-quality GPS data and microbiome profiles, exploratory individuals — measured by the number of spatially unique stops they made per day — carried significantly richer and more diverse microbiota, independent of capture site. This positive association held similarly at all three sites, implying that birds visiting more diverse locations sample their region more thoroughly and encounter more bacteria along the way. An alternative mechanism cannot be excluded: the microbiome itself might shape behavior, either through sickness or through deliberate host manipulation by microbial agents. Prior work in barn owls found that individuals moving greater distances from the nest had higher microbiome alpha diversity, and gull colonies using a wider variety of foraging sites showed maximal bacterial diversity, lending independent support to the exposure hypothesis.

The combined picture — high mobility, high densities, constant proximity to humans at roosts and cattle at foraging sites, and opportunistic visits to open habitats — points to a strong potential for feral pigeons to transfer pathogens among livestock, humans and wildlife. The authors emphasize that these patterns matter in both directions: pigeons are similarly likely to transmit pathogens from poultry farms into wildlife populations, a scenario underscored by the massive 2022 avian influenza outbreak. Practically, the findings suggest disease management should be tailored to local conditions and individual variation — culling protocols in rural areas would need to operate across much larger areas than urban ones, and more exploratory pigeons should be prioritized when targeting is possible. The study offers a rare mechanistic example of the One Health premise, demonstrating how movement ecology tools can illuminate the individual-level pathways through which synanthropic species connect human, animal and environmental health.

Subject of Research: Association between movement patterns, microbiome diversity, and potential pathogens of feral pigeons in dairy farms

Article Title: Association between movement patterns, microbiome diversity, and potential pathogens of feral pigeons in dairy farms

Article References: Crafton, M. M., Lublin, A., Cahani, S., Rauer, L., & Spiegel, O. (2025). Association between movement patterns, microbiome diversity, and potential pathogens of feral pigeons in dairy farms. Individual-based Ecology, 1, Article e146376. https://doi.org/10.3897/ibe.1.146376

Image Credits: AI Generated

DOI: 10.3897/ibe.1.146376

Keywords: Association, between, movement, patterns, microbiome, diversity, potential, pathogens, feral, pigeons, dairy, farms

The scientific rationale behind drSMALL rests on a decade of work showing that the microbes living in and around plants, the so-called symbiotic microbiota, can actively modulate how well their hosts resist pathogens. These microbes are prolific producers of secondary metabolites, a chemically diverse arsenal of small molecules that includes antibiotics, volatile organic compounds, quorum-sensing signals and phytohormone-like compounds. Some of these molecules directly suppress pathogens, while others prime the plant’s own immune system, triggering systemic resistance that protects tissues far from the site of colonization. Landmark studies cited by the authors include work showing that bacterial seed endophytes shape disease resistance in rice, that endophytic root microbiomes can be activated into disease-suppressive states by pathogens themselves, and that wheat microbiome bacteria can reduce the virulence of a fungal pathogen by altering histone acetylation in the fungus.

Despite this rich literature, the authors note that retrieving critical information remains slow and fragmented. Because both the symbiotic microbiota and their host plants are enormously diverse, and because the functions of small molecules are often context-dependent, even contemporary artificial intelligence tools struggle to make accurate predictions in this space. Researchers interested in whether a particular compound has been shown to confer disease resistance on a particular crop previously had to comb through scattered publications, often spending substantial time on retrieval. drSMALL addresses this by offering a centralized, web-based platform built on manually curated, evidence-based datasets extracted from the scientific literature, a distinction the authors emphasize because only molecules with experimental evidence linking them to plant disease resistance or disease suppression were admitted.

The data collection process was systematic. The team searched SciFinder, the Chemical Abstracts Service research discovery tool, using combinations of keywords pairing microbial taxa such as bacteria and fungi with terms for small molecules such as secondary metabolites and natural organic compounds, along with plant and disease resistance terms, covering literature up to October 2024. Each entry in the resulting database interlinks four kinds of information: the microbial species capable of producing the molecule, the small molecule itself, the host plant harboring the microbe, and the pathogen or disease that the molecule helps counter. Host plants in the database span a wide taxonomic range, including the grass family Poaceae, which contains rice, wheat and other cereals, as well as Solanaceae, Piperaceae, Cucurbitaceae, Brassicaceae, Rutaceae and Fabaceae, among others.

On the technical side, drSMALL is implemented as a web application designed for interactive visualization and rapid data retrieval. The front end is built on the Vue.js framework, which enables dynamic, user-friendly interfaces for exploring the data, while the back end runs on the Spring Boot framework, handling data collection, processing and management. Storage and retrieval rely on MySQL, an open-source relational database management system. The architecture reflects a deliberate choice to keep the platform lightweight and accessible: the database is open access, and the team commits to incorporating new relevant research findings promptly as they appear, while inviting researchers at the cutting edge of the field to submit additional data through a dedicated submission interface.

The user-facing design is organized around three core functionalities: Query, Classification Navigation and Submission. A search box at the top of the homepage allows users to type keywords related to plants, microbes, molecules or pathogens and quickly locate datasets of interest. Below the search box, a brief introduction outlines the features and content of the database. A central graphic illustrates the relationships among the resident microbiota, the molecules they produce, the host plants and the targeted pathogens, making the conceptual model of the resource immediately visible. On the right side of the page, four category options, plants, microbes, molecules and pathogens, lead to more detailed query interfaces, and an About page explains the platform’s features in greater depth.

The submission interface deserves particular attention because it turns drSMALL into a community resource rather than a static catalog. Users can contribute information about microbes, molecules and their CAS registry numbers, molecular formulas, host plants, targeted pathogens or diseases, and the relevant references, allowing the database to grow in step with the literature. The authors also provide a contact page for questions. This continuously updated, open-access model, they argue, is what will allow drSMALL to serve as a durable information hub for the plant-microbe interactions community, supporting both fundamental research into the chemistry of plant-microbe-pathogen tripartite interactions and applied work aimed at deploying beneficial microbes or their metabolites in the field.

Beyond its role as a lookup tool, the team sees drSMALL as a screening library for a much larger question: what else can these molecules do? Recent research has concentrated on two mechanisms, activation of plant immune responses and direct targeting of pathogens, but the potential of microbial small molecules to modulate other biological processes in plants remains largely unexplored. Whether the same compounds that confer disease resistance also promote growth, regulate metabolism, or enhance tolerance to biotic and abiotic stresses is a significant knowledge gap. Because the database aggregates molecules with proven disease-resistance activity alongside their producing microbes and host contexts, the authors suggest it can serve as a starting point for hunting hidden multi-bioactivities, a prospect with obvious appeal for breeding and biocontrol programs that want a single intervention to deliver multiple benefits.

The database also surfaces patterns that hint at where the field should look next. Some microbial species in the collection are notable for their capacity to secrete a variety of small molecules, marking them as potential reservoirs of bioactive chemistry worth deeper exploration. At the same time, the authors acknowledge uneven coverage: data on symbiotic microbes and their plant hosts remain limited compared with the extensive information available on pathogens and their hosts, and insufficiently studied crops such as sorghum and radish need further attention and updating. Closing those gaps, they write, will require contributions from researchers working on underexplored host plants and their resident microbiota, which is precisely what the submission pathway is designed to encourage.

Taken together, the launch of drSMALL reflects a broader shift in agricultural science away from a purely chemical model of crop protection and toward one that leverages the natural capabilities of plant microbiomes. By integrating and systematizing data on microbial species, small molecules and host plants, the platform gives researchers a rapid pathway to identify candidate molecules for studying previously unrecognized ecological and biological roles, and it lays a foundation for screening microbial chemistry with an eye toward multifunctionality. The authors frame the ultimate goal in terms of sustainable agricultural production and global food security: if the molecules that beneficial microbes use to shield plants from disease can be understood, cataloged and eventually deployed, whether as engineered microbiomes, microbial inoculants or nature-inspired crop treatments, they could help reduce reliance on synthetic agrochemicals whose overuse drives pathogen resistance and environmental contamination. drSMALL, freely available and open to community contributions, is presented as the first web-based resource to systematically organize this chemical dimension of plant disease resistance, and its value will grow with every study the community adds.

Subject of Research: A curated database of microbial small molecules experimentally linked to plant disease resistance

Article Title: drSMALL: Database for disease resistance-shaping small molecules derived from the plant microbiome

Article References: Cheng, R., Ke, T., Gui, F., Li, J., Zhang, X., Vílchez, J. I., & Matsumoto, H. (2025). drSMALL: Database for disease resistance-shaping small molecules derived from the plant microbiome. Crop Health, 3(1), Article 2. https://doi.org/10.1007/s44297-025-00042-7

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00042-7

Keywords: plant microbiome, small molecules, disease resistance, drSMALL, database, secondary metabolites, biocontrol, sustainable agriculture, crop health, plant-microbe interactions, pathogens, open access

The agricultural case for sludge is genuinely strong when the material is clean. Field studies cited in the review show that sludge application can raise crop yields by as much as 60 percent, rivaling the effect of fertilizers made from non-renewable resources. Two years of sludge amendment increased not only soil organic matter but also the stability of soil aggregates, improving soil structure in ways that conventional mineral fertilizers cannot. Biosolids even boosted the phenolic content and antioxidant activity of sweet basil, hinting at quality benefits beyond sheer yield. In a world facing dwindling phosphorus reserves and a nitrogen fertilizer crisis, the logic of recycling nutrients from human waste back into farmland is compelling. Circular-economy advocates argue that every tonne of sludge diverted to agriculture is a tonne of mined phosphate rock that does not need to be extracted.

The problem is what else rides along with those nutrients. Heavy metals, including cadmium, zinc, copper, chromium, nickel, arsenic, silver, and lead, make up roughly 0.5 to 2.0 percent of sludge on a dry-weight basis, and they originate from both industrial discharges and everyday household plumbing. Once these metals enter soil they are not biodegradable and can persist in the environment for decades, migrating through soil profiles into groundwater and accumulating in the edible tissues of crops. One influential study found that while sludge boosted yields of mung bean by up to 60 percent, it simultaneously raised the accumulation of lead, nickel, and cadmium in the grain, undermining its suitability as food. Under European rules for organic-mineral fertilizers, permissible limits are 100 milligrams of chromium, 5 milligrams of cadmium, 60 milligrams of nickel, 140 milligrams of lead, and 2 milligrams of mercury per kilogram, but the actual metal content of a given sludge depends heavily on its origin, and the same production technology can deliver strikingly different results depending on whether the process runs at laboratory, pilot, or full industrial scale.

Pathogens constitute a second, more insidious barrier. Municipal sludge is a biological concentrate of fecal bacteria such as Escherichia coli and Salmonella, enteric viruses including rotavirus, norovirus, and hepatitis A, protozoan parasites like Giardia intestinalis, and fungal genera such as Aspergillus and Penicillium. Farmers handling sludge-based fertilizers report skin allergies, digestive ailments, and respiratory infections, and communities living near fields where sludge is sprayed face elevated risks of pharyngitis, coughing, and shortness of breath from airborne transmission. Contaminated soil can also shed pathogens into surface water and groundwater during rainfall, threatening drinking supplies and aquatic biodiversity. The review stresses a subtle analytical problem: pathogen counts measured with culture-based methods versus molecular assays such as quantitative PCR are not directly comparable, and detecting viral genetic material does not necessarily mean the virus is still infectious. Comparisons across studies are further muddied by differences in sludge stabilization history, storage conditions, and seasonal variation in the incoming wastewater.

Organic micropollutants form perhaps the most chemically diverse contaminant group. The most frequently detected compounds include linear alkylbenzenesulfonates from detergents, the plasticizer di-ethylhexylphthalate, nonylphenols, polychlorinated biphenyls, dioxins and furans, and polycyclic aromatic hydrocarbons, which have been measured at concentrations ranging from 0.7 to 75.2 milligrams per kilogram in sludge. Persistent compounds such as DEHP and PAHs are of particular concern because they have been linked to cancer, reproductive damage, and metabolic disease. Pharmaceuticals add another layer: global antibiotic consumption rose 65 percent between 2000 and 2015, and most antibiotics pass through the human body unmetabolized, ending up in sludge at concentrations from micrograms to milligrams per kilogram. When antibiotic-laden sludge is applied to soil, it exerts selective pressure that fosters antibiotic-resistant bacteria, suppresses microbial diversity, and can inhibit carbon mineralization, nitrogen cycling, and enzyme activity. Natural and synthetic steroid hormones, active at vanishingly low concentrations, raise endocrine-disruption worries, while persistent pesticides accumulate in sludge solids because of their hydrophobic chemistry.

Microplastics have emerged as the contaminant that most sharply exposes the limits of current treatment and regulation. An estimated 3.2 million tonnes of primary microplastics enter the environment annually, and between 60 and 99.9 percent of the microplastics entering wastewater treatment plants are retained in the resulting biosolids. Studies report anywhere from hundreds of thousands to several millions of microplastic particles per kilogram of sludge, depending on the analytical method and particle-size range considered. Once sludge is spread on fields, these particles, which can adsorb dyes, heavy metals, and pharmaceuticals onto their surfaces, effectively seed agricultural soils with a persistent plastic burden and a mobile carrier for co-transported pollutants. The United Nations listed microplastic pollution among the top ten environmental issues requiring immediate attention in 2017, yet quality standards for biosolids and soils, many written three decades ago, still test only for heavy metals and a handful of persistent organic pollutants. Microplastics, pharmaceuticals, hormones, and antibiotic resistance determinants fall almost entirely outside the regulatory net.

The review catalogues a battery of treatment technologies that can shrink these risks, while candidly admitting that none is a universal solution. For heavy metals, options include alkaline modification with geopolymers that immobilize metals in monolithic matrices, chemical extraction using chelating agents such as EDTA and NTA followed by sulfide precipitation, microbiological bioleaching with organisms like Acidithiobacillus ferrooxidans that achieved up to 39 percent lead removal in one study, and electrokinetic treatment that drives metal ions toward electrodes under a direct-current field. Each approach carries trade-offs: stabilization does not remove total metal mass and can reverse if soil pH or redox conditions shift, extraction generates secondary metal-rich effluents, bioleaching is slow and finicky, and electroreclamation is energy-hungry and electrode-sensitive. For pathogens, composting under controlled conditions eliminated 99 percent of Salmonella and 96 percent of fecal coliforms within a month, while thermophilic anaerobic digestion at 50 to 60 degrees Celsius denatures bacterial enzymes and outperforms mesophilic digestion. Quicklime treatment pushes pH above 12 and heats sludge to around 70 degrees, destroying organisms including Clostridium perfringens, Listeria, and Enterococcus, and hybrid schemes combining 160-degree thermal hydrolysis with anaerobic digestion came close to complete pathogen destruction, sparing only Clostridium perfringens. Irradiation with cobalt-60 or cesium-137 can shred pathogen DNA but remains rare at full scale because of cost and safety barriers.

For organic micropollutants, well-managed composting reduced hormones below detection limits in field-scale post-treatment, whereas uncontrolled open-air storage produced variable and often disappointing removal. Anaerobic digestion shows compound-specific performance, achieving over 80 percent removal for selected anti-inflammatory drugs under laboratory conditions but low or even negative removal for other pharmaceutical classes at full scale. Thermal routes are the heavy artillery: combustion at 850 to 1000 degrees destroys most organic micropollutants but transfers a fraction of them to flue-gas residues, and pyrolysis at 600 degrees reduced polychlorinated biphenyls, PAHs, and pharmaceutical and personal-care compounds by at least 99.9 percent in one comparative study, although contaminants can partition into condensates and oils that then require controlled disposal. Against microplastics, incineration removes roughly 99.9 percent of particles and pyrolysis achieves 91 to 97 percent removal, but both funnel nutrients out of closed-loop recycling into construction materials or energy, while emerging alternatives such as hydrothermal liquefaction and hyperthermophilic composting show promise yet rarely exceed 50 percent removal efficiency and remain poorly characterized in terms of degradation by-products.

The review’s most sobering findings concern the law rather than the lab. Council Directive 86/278/EEC protects soils by setting heavy-metal thresholds for agricultural sludge, but the EU fertilizer framework, Regulation 2019/1009, excludes sludge from its list of approved fertilizer ingredients precisely because of toxicity concerns, leaving farmers who wish to use it dependent on temporary national permits. Because member states write their own rules on application rates, permissible microorganism levels, microplastics, antibiotics, hormones, and pH, soil protection is inconsistent across the bloc, and internationally traded food faces uneven safety standards. The authors call for a harmonized directive specifying contaminant limits across all pollutant classes, mandatory annual soil testing for heavy metals, microplastics, hormones, and antibiotics, adoption of best available techniques, and on-site sludge processing to cut transport emissions. Public skepticism, fueled by odors and documented health complaints near land-application sites, adds a social dimension that educational campaigns and community engagement must address. The bottom line is pragmatic: sludge-derived fertilizers can genuinely anchor circular nutrient management in a phosphorus-scarce world, but only under science-based regulation, standardized analytical methods, and treatment strategies that account for contaminant fate, secondary waste streams, and real-world field conditions rather than laboratory promises alone.

Subject of Research: Contaminant risks and mitigation strategies in sewage sludge-derived fertilizers

Article Title: Assessing and Mitigating Risks of Emerging Contaminants in Fertilizer Production from Sewage Sludge: Challenges and Opportunities

Article References: Skrzypczak, D., Mikula, K., Izydorczyk, G., Samarina, T., Gil, F., Wijatkowska, A., Szyszka, D., & Chojnacka, K. (2026). Assessing and Mitigating Risks of Emerging Contaminants in Fertilizer Production from Sewage Sludge: Challenges and Opportunities. Waste and Biomass Valorization. https://doi.org/10.1007/s12649-026-03780-z

Image Credits: AI Generated

DOI: 10.1007/s12649-026-03780-z

Keywords: sewage sludge, heavy metals, pathogens, microplastics, pharmaceuticals, composting, anaerobic digestion, pyrolysis, fertilizer production, circular economy, wastewater treatment, regulatory gaps