Pathogenic bacteria deploy biofilm as a key virulence factor to cause plant vascular diseases, which are devastating to global agricultural practices. Extracellular DNA (eDNA) constitutes the backbone of bacterial biofilm and is key to biofilm stability, thereby representing as an attractive therapeutic target. Here, we engineered the plant chloroplast-localized Holliday junction (HJ) resolvase MOC1 by replacing its native chloroplast transit peptide with a secretory signal, successfully relocating it to the apoplast. Transgenic tomato and rice expressing secreted MOC1 exhibited robust resistance to bacterial wilt and bacterial blight, respectively, without growth or yield penalties. Additionally, we implemented bacterial pathogen-inducible promoters to achieve precisely spatial and temporal control over the resistance trait. Secreted MOC1 degrades eDNA in situ, disrupts biofilm architecture, and markedly reduces bacterial colonization and systemic spread. Our work presents a novel strategy for controlling vascular diseases by engineering plant HJ resolvases to disrupt biofilms. This approach provides a new blueprint for molecular resistance breeding and disease resistance gene exploration.

Mycoviruses are typically transmitted vertically through fungal reproduction or horizontally via hyphal anastomosis, but identical viruses in phylogenetically divergent fungi hint at unknown inter-species transmission mechanisms. Here, we identify fungal extracellular vesicles (EVs) as mediators of cross-genus mycovirus transmission. Using the hypovirulent Botrytis cinerea strain IBc-374 (harboring 16 mycoviruses) as a donor, we show that up to 13 mycoviruses are horizontally transmitted to Sclerotinia sclerotiorum during dual culture or plant co-inoculation, even though these two fungi belong to different genera and are generally considered incapable of hyphal anastomosis. Electron microscopy reveals abundant vesicle structures in IBc-374 hyphae, and nanoparticle tracking analysis showed that the strain secretes >11-fold more EVs than a virus-free strain. RT-PCR detects genomic RNAs of 7 mycoviruses in purified EVs, and the full-length viral genome in EVs was further validated with BcHV5 as example by fluorescence in situ hybridization and RT-PCR. Incubation of protoplast-derived germlings of S. sclerotiorum or B. cinerea with IBc-374 EVs leads to infection by 4-6 donor mycoviruses, demonstrating EV-mediated cross-genus transmission. Injection of mycovirus-carrying EVs into tobacco leaves followed by fungal inoculation also transmits two hypoviruses to both species. Application of IBc-374 hyphal fragment suspension significantly reduces lesion sizes caused by both pathogens on plants, and rescued two pathogens carrying multiple mycoviruses exhibit hypovirulence and impaired growth. Our findings reveal EVs as a cell-free vector that bypasses vegetative incompatibility, providing a mechanistic basis for cross-species viral spread and opening avenues for EV-based virus cocktails to control multiple fungal diseases.

Clubroot, caused by the protist pathogen Plasmodiophora brassicae, is a major threat to cruciferous crop production worldwide. Although plant microbiota is known to influence disease outcomes, the mechanisms underlying microbiota-mediated resistance remain unclear. Here, we investigated the role of plant microbiota in clubroot resistance using two Chinese rapeseed cultivars carrying resistance genes introduced through breeding and their susceptible parental lines. Microbiome profiling revealed that P. brassicae infection altered root and rhizosphere bacterial communities, with resistant cultivars displaying distinct assemblages. Functional prediction indicated an enrichment of denitrifying bacteria in the roots of resistant plants following pathogen challenge. Key denitrifying strains were isolated and assembled into a synthetic microbial community (SynCom18), which significantly suppressed clubroot development under both controlled and field conditions. In addition to reducing disease severity, microbial treatments improved agronomic traits, including yield and seed quality. Mechanistic analysis revealed a positive correlation between soil nitrate levels and disease severity. Denitrifying strains and SynCom18 likely suppressed the development of P. brassicae and enhanced plant immunity by reducing soil nitrate levels by ~39.4%. Metabolomic profiling revealed that aesculetin, as a dominant metabolite that is produced by resistance roots and excreted into the rhizosphere to recruit denitrifying bacteria. Our findings show that pathogen-infected clubroot-resistant rapeseed cultivars secrete aesculetin to recruit nitrate-depleting bacteria for resistance against P. brassicae. This study elucidates a tripartite microbiota-pathogen-soil nutrient interaction and provides a sustainable biocontrol strategy for cruciferous crops.

In plants, reactive oxygen species (ROS) play a crucial role in rapidly responding to biotic stresses, thus contributing to the establishment of immune networks and plant resistance against pathogen attack. The two-layered plant immune system consists of the cell-surface pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and intracellular effector-triggered immunity (ETI), both of which are associated with ROS burst. Mitochondria, serving as a major source of intracellular ROS, are key to plant immunity, and their function depends on the RNA processing of mitochondrial genes. The DEAD-box RNA helicase PUTATIVE MITOCHONDRIAL RNA HELICASE 2 (PMH2) is required for efficient group Ⅱ intron splicing in mitochondria; however, how PMH2-mediated fine RNA splicing contributes to plant immunity remains unknown. Here, we revealed a function of PMH2 in ETI using Arabidopsis thaliana. ETI activation led to the PMH2-mediated reduction in splicing efficiency of cox2, which encodes the subunit of mitochondrial respiratory chain complex IV, and the activity of complex IV, thereby promoting the generation of mtROS. Moreover, PMH2-mediated mtROS facilitated the expression of the nuclear immunity genes. These results collectively suggest that PMH2 contributes to specific mitochondrial RNA splicing and fine-tunes the mitochondrial ROS burst, therefore maintaining robust plant immunity. Our study provides the mechanism of RNA helicase linking RNA processing and mitochondrial dynamics to plant ETI.

In addition to wound-related infections such as green mold, blue mold, and sour rot, latent infections represent the most common type of postharvest disease in citrus fruits (Citrus unshiu). These diseases are particularly difficult to manage, and their underlying pathogenic mechanisms remain poorly characterized. In this study, we used ITS sequencing to monitor the dynamics of microbial communities in citrus fruits from preharvest development through storage. Analysis of beta diversity revealed significant changes in fungal community composition. Notably, greater variability was observed across preharvest developmental stages, whereas postharvest samples showed relatively limited variation. Microbial interaction network and LEfSe analyses further indicated more stable microbial network in preharvest samples. Additionally, a marked reduction in the abundance of core microorganisms was observed during storage compared to pre-harvest stages. Investigations of postharvest disease showed that rot caused by fungi of genera Alternaria, Fusarium, and Colletotrichum was the predominant diseases, with the disease incidence showing a positive correlation with the abundance of Alternaria and Colletotrichum. At the same time, the abundance of beneficial microorganisms declined significantly after harvest. Therefore, the decrease in beneficial microorganisms, coupled with the increase in potential pathogenic fungi, probably plays a major role in the occurrence of postharvest diseases in citrus fruits. This study provides a theoretical foundation for understanding the pathogenesis of latent infections and offers new perspectives for controlling these diseases.

China is one of the largest kiwifruit-growing countries in the world. In recent years, post-harvest decay of kiwifruit has occurred widely in major cultivation areas, severely affecting the development of Chinese kiwifruit industry. Our previous study indicated that Diaporthe species were one of the dominant pathogens of the decay in China. In this study, further research was conducted on the diversity and pathogenicity of pathogens within the genus. Based on ITS sequences, 40 representative isolates were selected from 325 Diaporthe fungal isolates. Through the combination of multi-locus phylogenetic analysis and morphological observations, the 40 isolates were identified as 8 species, including four known pathogenic species: Diaporthe eres, Diaporthe sojae, Diaporthe hongkongensis, and Diaporthe passiflorae and four new host species: Diaporthe arecae, Diaporthe amygdali, Diaporthe tulliensis, and Diaporthe apiculata. Among the 325 isolates, D. eres had the highest abundance (58.9%) and the widest distribution (Jiangxi, Hubei, Shaanxi, Sichuan, and Shandong provinces). D. hongkongensis followed with a percentage of 3.7% and was distributed in Jiangxi, Hubei, and Shaanxi provinces. Pathogenicity tests showed all eight species infected both kiwifruit (Hayward) and apple (Tiaohong), with D. amygdali and D. hongkongensis exhititing the highest virulence on kiwifruit and D. arecae and D. hongkongensis on apple. Six species cross-infected citrus (Satsuma mandarin), notably D. tulliensis and D. arecae. Additionally, the eight species also exhibited varying degrees of pathogenicity on kiwifruit shoots. This study not only contributes to the understanding of the etiology of kiwifruit decay in China but also enriches the knowledge of fungal diversity within the Diaporthe genus, providing crucial insights for developing integrated management strategies against these cross-infectious pathogens.

Hypovirulence-associated mycoviruses can be frequently isolated in nature despite their likely compromised ecological fitness, but how their fungal hosts survive in natural environments remains largely unresolved. The discovery of more mycoviruses provides opportunities to understand these mycovirus-host relationships. Here, we characterize a capsidless RNA virus, Sclerotinia sclerotiorum ascoshuvirus 1 (SsAShV1), with a 10.7--kb ssRNA genome encoding a polyprotein containing conserved protease domain and RNA--dependent RNA polymerase (RdRP) domain. SsAShV1 shares structural similarities with animal--infecting viruses. SsAShV1 and its phylogenetically related shuviruses form a distinct evolutionary lineage, prompting the proposal to establish the family Shuviridae. SsAShV1 transfection alone induces hypovirulence in two Sclerotiniaceae fungal pathogens, with its 3'-UTR repeat--containing structural region (RCSR) enhancing replication efficiency. In the hypovirulent strain SCH767, which harbors SsAShV1 and five other mycoviruses, we observed a synergistic enhancement of antifungal volatile organic compound (VOC) production that was dependent on the host genetic background. A VOC, 2-ethyl-1-hexanol, exhibited broad-spectrum antimicrobial activity. Our findings reveal an evolutionary trade-off where viral infection shifts the fungal survival strategy from high virulence to enhanced chemical competition, ensuring the persistence of the hypovirulent partnership, and offering valuable insights into the development of a combination of mycovirus-based and VOC-mediated biocontrol strategies.

Sclerotinia sclerotiorum is a destructive pathogen with a broad host range, long-term soil survival, and is difficult to control. Silencing virulence-related genes is a strategy for controlling Sclerotinia disease. In this study, we identified and characterised Sspdhx, which encodes pyruvate dehydrogenase complex component X in S. sclerotiorum. Sspdhx deletion exhibited significant impairments in growth, sclerotia development, infection cushion formation, and virulence, indicating that Sspdhx plays important biological functions in S. sclerotiorum. Sspdhx deletion also resulted in reducing acetyl-CoA and ATP levels, and increased sensitivity to multiple environmental stresses. Exogenous supplementation with acetyl-L-carnitine partially restored the virulence of the ΔSspdhx mutants. Transcriptomic analyses revealed that deletion of Sspdhx disrupts central carbon metabolic homeostasis, leading to broad transcriptional reprogramming that affects genes involved in vegetative growth, stress adaptation, and virulence-associated processes. Application of exogenous Sspdhx-targeting dsRNA and host-induced gene silencing in plants effectively silenced Sspdhx and attenuated the virulence of S. sclerotiorum. These findings potentially establish Sspdhx as a promising target for RNA-based control strategies against Sclerotinia disease.

Abstract Crop soilborne diseases caused by pathogens, such as Fusarium spp. and root-knot nematode (RKN), contribute to substantial yield losses worldwide. This study investigated the biocontrol potential of the fungivorous nematode Aphelenchoides bicaudatus isolate A23 against soilborne diseases. Isolate A23 showed strong antagonism against phytopathogenic fungi, while remaining non-pathogenic to tomato plants. Among the four soilborne pathogenic fungi tested, A. bicaudatus exhibited a pronounced feeding preference for Fusarium spp., reducing the incidence of tomato Fusarium wilt by up to 60%. Moreover, in both pot and field trials, A. bicaudatus significantly suppressed root-knot nematode attack, reflected in reduced galling index, egg masses, and egg production, thereby increasing fruit yield relative to the control. Split-root assays and qRT-PCR analysis demonstrated that root-knot nematode suppression by A. bicaudatus was primarily mediated by induced systemic resistance, involving the activation of salicylic acid-dependent defense pathways. Additionally, high-throughput amplicon sequencing indicated that A. bicaudatus inoculation reshaped the rhizosphere microbiome by enriching beneficial bacterial and fungal genera while reducing pathogen-associated taxa, such as Fusarium . Collectively, these results demonstrate that the A . bicaudatus isolate A23 is an effective biocontrol agent that acts through an integrated mechanism combining direct fungal predation, plant defense priming, and beneficial microbiome reshaping. This study establishes a foundation for using fungivorous nematodes in a multifunctional role in crop disease management.

A new double-stranded RNA mycovirus, tentatively named "Sclerotinia sclerotiorum victorivirus 2" (SsVV2), was isolated from Sclerotinia sclerotiorum strain DT-110. The complete genome of SsVV2 is 5135 nucleotides in length and contains two open reading frames (ORF1 and ORF2) that overlap at the tetranucleotide AUGA (positions 2546-2549). ORF1 and ORF2 were predicted to encode a coat protein (CP, 755 amino acids) and an RNA-dependent RNA polymerase (RdRP, 836 amino acids), respectively. BLASTp analysis identified Sclerotinia nivalis victorivirus 1 (SnVV1) as the closest match to SsVV2, with their RdRPs sharing 75.7% amino acid sequence identity. Phylogenetic analysis based on the RdRP and CP of victoriviruses further support the clustering of SsVV2 with SnVV1. These results confirm that SsVV2 as a new member of the species Victorivirus nijyusani, genus Victorivirus, family Pseudototiviridae.

Mycoviruses are ubiquitous in fungi, and some of these viruses induce hypovirulence, making them potential biocontrol agents. However, the effect of host genetic background at the pathogen population level on the performance of mycoviruses has not been fully investigated. Here, five diverse species of mycoviruses in Botrytis cinerea were used as a model to investigate the effect of population variation on mycovirus-induced phenotypes. The five mycoviruses were fully sequenced, including two hypoviruses (dsRNA1 and dsRNA2), one victorivirus (dsRNA3), and one partitivirus (dsRNA7 and dsRNA8), while one virus (dsRNA4, dsRNA5, and dsRNA6) remained unclassified. These viruses were readily transmitted to three B. cinerea strains, resulting in several derivative strains infected by different combinations of the five viruses. Notably, infection by some of these five viruses generally increased sporulation. However, the symptoms induced by the viruses varied, ranging from no significant effects to reduced virulence among different B. cinerea strains with the same viral profile. Therefore, we propose that the B. cinerea population can be broadly categorized into sensitive and insensitive groups regarding viral infection. These findings suggest that variation within the B. cinerea population may affect mycovirus-mediated phenotypic changes in their hosts. The presence of the insensitive group within the fungal population could also complicate the application of mycoviruses for fungal disease control.

BACKGROUND:The Hda1 protein, a conserved Class II histone deacetylase, plays a key role in deacetylating histone proteins and regulating gene transcription. Although its importance has been established in various organisms, the functional role of Hda1 in insect pathogenic fungi has not been thoroughly investigated. RESULTS:In this study, we explore the function of BbHda1 in the entomopathogenic fungus Beauveria bassiana. The absence of BbHda1 dramatically enhanced bulk acetylation of histone H4, but had no observable effect on histone H3 acetylation. Consequently, the deletion of BbHda1 led to several notable phenotypic changes, including decreased conidial yield, slowed conidial germination and impaired conidial properties. Additionally, the ΔBbHda1 mutant exhibited reduced resistance to multiple stresses, such as oxidative stress, cell wall perturbation and DNA damage. Notably, the ΔBbHda1 mutant displayed significantly slower growth in insect hemocoel and reduced virulence. Comparative transcriptomic analysis revealed a wide array of downregulated genes involved in various biological processes, including secondary metabolism, chemoperception and response, detoxification involving cytochrome P450, and multiple drug transporters and virulence factors. Our findings revealed that BbHda1 is essential for modulating sporulation, multistress resistance and fungal virulence, primarily through its control of histone acetylation and transcriptional regulation. CONCLUSION:These results elucidated the critical role of BbHda1 in development, stress response, and virulence in B. bassiana, and provide valuable insights into the post-translational mechanisms underlying fungal pathogenesis. © 2025 Society of Chemical Industry.

Mycoviruses are increasingly recognized for their multifaceted roles in fungal ecology, because of advances in understanding of their biology and molecular features. In this research, we identified and characterized two capsidless, bi-segmented positive-sense RNA mycoviruses: Verticillium dahliae ormycovirus 1 (VdOMV1) and VdOMV2, both of which infect Verticillium dahliae, a fungal pathogen causing vascular wilt of cotton. Phylogenetic analysis revealed that VdOMV1 and VdOMV2 cluster within the ormycovirus group, an evolutionary lineage unique to Riboviria. VdOMV2 may significantly enhanced V. dahliae melanin production and microsclerotial formation through regulating melanin synthesis-associated genes. This mediated the conversion from production of hyphae to microsclerotia, and enhanced V. dahliae survival under adverse abiotic stress conditions. Furthermore, VdOMV2 boosted the penetration ability of hyphae through cellophane membranes, while inhibiting the proliferation of V. dahliae hyphae within plants, and negatively modulated genes related to pathogenicity, possibly conferring hypovirulence. Enhancements in penetration and survival not only increase the efficacy of hypovirulent strains in overcoming environmental challenges, but also highlight the potential of VdOMV2-infected strains for managing Verticillium wilt in agricultural settings, thus representing an alternative mycovirus-based biocontrol approach for vascular fungal diseases.

Plants perceive microbe-derived molecular patterns to initiate the innate immune system. The csp22 peptide, derived from bacterial cold shock protein, is uniquely recognized by cold shock protein receptor (CORE) in Solanaceae plants, yet the signaling pathway remains largely obscure. In this study, we identify that tomato csp22-activated kinase 1 (SlCAK1), belonging to the receptor-like cytoplasmic kinase subgroup VII, is essential for csp22-induced immune responses and resistance to bacterial wilt disease. SlCAK1 is rapidly recruited to the csp22 receptor complex, which results in SlCAK1 phosphorylation upon csp22 treatment. Notably, csp22-induced phosphorylation specifically occurs at the Ser60 residue in the N terminus of SlCAK1, which is critical for SlCAK1 function in immunity. Interestingly, the conservation of Ser60 is unique to Solanaceae plants. Taken together, our findings reveal the pivotal role of SlCAK1 in transducing csp22-triggered immune signaling and provide a novel activation mechanism for receptor-like cytoplasmic kinases that involves phosphorylation in the N terminus.

Bacterial wilt caused by Ralstonia solanacearum is a devastating plant disease. Exopolysaccharide (EPS), a major virulence factor of R. solanacearum, elicits pattern-triggered immunity (PTI) in tomato, but the means by which EPS is recognized in the plant remain poorly understood. We found that tomato non-arginine-aspartate (non-RD) receptor kinase SlLYK4 mediates the perception of R. solanacearum EPS and positively regulates resistance to bacterial wilt. The RD receptor kinases SlLYK1 and SlLYK13 are required for EPS-triggered immune responses and form complexes with SlLYK4. These receptor kinase complexes have dual functions in recognizing bacterial EPS and fungal chitin. Phosphorylation of serine-320 in the juxtamembrane domain of SlLYK4 is essential in EPS- and chitin-mediated signaling, whereas phosphorylation of serine-334 or serine-634 in the C-terminal domain is required for chitin or EPS signaling, respectively. Our results reveal the mechanism underlying EPS recognition in tomato and provide insight into how differential phosphorylation of receptor kinase regulates antibacterial and antifungal immunity.

Sclerotinia sclerotiorum, known as a typical necrotrophic pathogenic fungus, exhibits a complex pathogenic mechanism. Research on S. sclerotiorum has primarily focused on oxalic acid, pathogenicity-related enzymes, and secreted proteins. In this study, we identified a transcription factor, SsSR (S. sclerotiorum Sterol-Related transcription factor), which regulates S. sclerotiorum infection by modulating virulence through ergosterol biosynthesis. We characterized the transcriptional activity of SsSR and its downstream target gene, SsCYP51. SsSR undergoes phosphorylation induced by the host plant, subsequently regulating the expression of SsCYP51. The deletion of SsSR or SsCYP51 does not affect the growth or acid production of S. sclerotiorum, but it leads to a reduction in ergosterol, significantly diminishing virulence and impairing the stress tolerance of the hyphae. In summary, this study identifies a transcription factor, SsSR, that specifically regulates the virulence of S. sclerotiorum. SsSR upregulates the expression of SsCYP51 through phosphorylation during the infection phase, leading to the synthesis of ergosterol, which enhances hyphal stress tolerance and thereby promotes infection.

Sclerotinia sclerotiorum, a fungal pathogen that is spread worldwide and causes serious diseases on crops, can be parasitized specifically by the mycoparasite Coniothyrium minitans. SsNEP2, encoding a necrosis-inducing protein in S. sclerotiorum, was previously inferred to play a role in the virulence to host plants. In this study, silencing of SsNEP2 in S. sclerotiorum had no significant (p < 0.01) influence on mycelial morphology, while overexpression led to lower mycelial growth and more branches. When amended with the fermentation broth of the SsNEP2 silencing mutants, conidial germination of C. minitans was promoted, while conidial production decreased. When parasitized by C. minitans, enhanced resistance of the SsNEP2 silencing mutants and weaker resistance of the overexpressed transformants were observed compared to the wild-type S. sclerotiorum strain 1980. In addition, the expression of SsNEP2 in C. minitans enhanced mycelial parasitism on S. sclerotiorum and restored the effect of silencing SsNEP2 in S. sclerotiorum on mycoparasitism. Thus, we highlight the role of SsNEP2 as a PAMP-like protein in the mycoparasitism between C. minitans and its host fungus S. sclerotiorum. SsNEP2 can be used to promote the biological potential of C. minitans.

Sclerotinia sclerotiorum is a worldwide plant pathogenic fungus. Identifying novel mycoviruses in this fungus can aid in developing fungal disease control strategies and enhance our understanding of viral evolution. Here, we analyzed mycovirus composition in S. sclerotiorum strain XZ69, and identified six ssRNA mycoviruses, including five known mycoviruses and one unassigned mycovirus. The newly identified mycovirus, tentatively named Sclerotinia sclerotiorum narna-like virus 1 (SsNLV1/XZ69), possesses a full-length genome of 3534 nucleotides, containing a single ORF that encodes an RNA-dependent RNA polymerase (RdRp) of 1090 amino acids. The RdRp encoded by SsNLV1/XZ69 shares 60.4 % identity with that encoded by Monilinia narnavirus H. SsNLV1/XZ69 phylogenetically clusters with unclassified narna-like viruses potentially infecting fungi, plants, and animals, and they form an independent branch that is distant from established families, therefore supporting the establishment of a new family to accommodate these viruses. Sclerotinia sclerotiorum fusarivirus 3 (SsFV3/XZ69) share 97 % amino acid identities with preciously reported Botrytis cinerea fusarivirus 8 (BcFV8). This last mycovirus originated from Botrytis cinerea, and hence this reveals that cross-genus transmission of SsFV3 or BcFV8 between B. cinerea and S. sclerotiorum may have potentially occurred. Mycovirus elimination, horizontal transmission, and RNA transfection experiments revealed that Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV1/XZ69), SsNSRV2/XZ69, and SsFV3/XZ69 may be associated with hypovirulence in S. sclerotiorum, and strain XZ69 exhibits potential disease biocontrol on rapeseed seedlings. Our study expands our understanding of viral evolution, and may provide new potential biocontrol agents for S. sclerotiorum.

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.