Histopathological changes of Ceroplastes japonicus infected by Lecanicillium lecanii

The infection process and pathological changes of Japanese wax scale, Ceroplastes japonicus Green, by the hyphomycete Lecanicillium lecanii (Zimmermann) Gams & Zare were investigated by light, scanning and transmission electron microscopy. The results showed that L. lecanii generally infected the wax scale by penetrating the integument. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test had an inhibitory effect on L. lecanii. Within 24 h after inoculation, conidia became attached to the cuticle, and within 48 h, hyphae adhered to the integument of the scale and their tips differentiated into specialized infection pegs. Penetration of the cuticle occurred within 72 h of inoculation; the fungus caused the insect cuticle to rupture and hyphae entered the insect body through these openings. Within 72 h after inoculation, L. lecanii entered the hemocoele of the scale and formed blastospores. After 96 h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g., tracheae, malpighian tubules and muscle fibers, were also infected. As the infection progressed, the wax test and body changed color from white and red, respectively, to yellowish. After 144 h, the internal tissue structure was totally compromised and the insects died. After this time, new conidiophores bearing conidia were produced on the surface of the cadavers.     

Cellular encapsulation in the eastern subterranean termite, Reticulitermes flavipes (Isoptera), against infection by the entomopathogenic fungus Metarhizium anisopliae

Reticulitermes flavipes workers were topically inoculated with ≈10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-9 d, histopathological examination showed that termites had an individual immune reaction. The nodule formation at the point of entrance of the fungal hyphae was identified as a cellular encapsulation and the different steps in the nodule formation are described. The relative number of hemocytes per termite increased 24 h after fungal exposure and remained high in the hemolymph for at least 3 d before decreasing back to pre-exposure levels. The role of an individual immune cellular reaction in social insects is discussed.     

Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi

Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode''s cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 μmhos, 20 ± 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 μmhos) than in undiluted saturation extract (conductivity = 780 μmhos). Cat produced small zoospores (4-μm-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-μm-d) and tubular, uniform infection and assimilative hyphae in nematodes.     

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E₂ 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E₂ titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E₂ or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E₂ raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Ultrastructural analysis of the behavior of the dimorphic fungus Microbotryum violaceum in fungus-induced anthers of female Silene latifolia flowers

Summary. The development of male organs is induced in female flowers of the dioecious plant Silene latifolia by infection with the fungus Microbotryum violaceum. Stamens in a healthy female flower grow only to stage 6, whereas those in an infected female flower develop to the mature stage (stage 12), at which the stamens are filled with fungal teliospores instead of pollen grains. To investigate these host–parasite interactions, young floral buds and fungus-induced anthers of infected female flowers were examined by electron microscopy following fixation by a high-pressure freezing method. Using this approach, we found that parasitic hyphae of this fungus contain several extracellular vesicles and have a consistent appearance up to stage 8. At that stage, parasitic hyphae are observed adjacent to dying sporogenous cells in the infected female anther. At stage 9, an increased number of dead and dying sporogenous cells is observed, among which the sporogenous hyphae of the fungus develop and form initial teliospores. Several types of electron-dense material are present in proximity to some fungi at this stage. The initial teliospores contain two types of vacuoles, and the fungus cell wall contains abundant carbohydrate, as revealed by silver protein staining. The sporogenous cell is probably sensitive to infection by the fungus, resulting in disruption. In addition, the fungus accelerates cell death in the anther and utilizes constituents of the dead host cell to form the mature teliospore. Correspondence and reprints (present address): Molecular Membrane Biology Laboratory, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.     

Attraction of four entomopathogenic nematodes to four white grub species

To better understand the differences in the efficacy of entomopathogenic nematode species against white grub species, we are studying the various steps of the infection process of entomopathogenic nematodes into different white grub species using nematode species/strains with particular promise as white grub control agents. In this study we compared the attraction of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), Steinernema glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and Heterorhabditis bacteriophora (GPS11 strain) to third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis, and late-instar greater wax moth, Galleria mellonella, larvae. Individual larvae were confined at the bottom of 5.5 cm vertical sand columns, nematodes added to the sand surface after 24 h, and nematodes extracted after another 24 h. Nematode attraction to hosts was strongly affected by nematode species but the effect of insect species varied with nematode species. S. glaseri had a high innate dispersal rate (i.e., in absence of insects) and was strongly attracted to insects without significant differences among insect species. S. scarabaei had a very low innate dispersal rate so that even a strong relative response to insects resulted in low absolute dispersal rates toward insects. S. scarabaei tended to be most attracted to G. mellonella and least attracted to C. borealis. H. zealandica had a high innate dispersal rate but only responded weakly to insects without significant differences among species. H. bacteriophora had limited innate dispersal and only weakly responded to insects with G. mellonella tending to be the most attractive and C. borealis the least attractive insect. It has to be noted that we cannot exclude that the use of different rearing hosts (A. orientalis and P. japonica larvae for S. scarabaei, G. mellonella larvae for the other nematodes) might have had an impact on the nematodes dispersal and relative attraction behavior. This study indicates that host attractiveness and nematode dispersal rates may contribute but do not play a major role in the variability in white grub susceptibility and/or nematode virulence.     

Colonization of Soybean Cyst Nematode Females,Cysts, and Gelatinous Matrices by the Fungus Verticillium lecanii

Heterodera glycines was grown in monoxenic culture on soybean roots and then inoculated with the antagonistic fungus Verticillium lecanii. Use of root explant cultures allowed evaluation of the fungus-nematode interaction with the nematode attached to roots or removed from the host, and avoided contamination with other fungi. From 16 hours to 14 days following inoculation, female and cyst samples were examined with the light microscope, or prepared for either conventional or low-temperature scanning electron microscopy. Within 16 hours, hyphae had begun colonizing the gelatinous matrices (GM). The fungus proliferated in the GM of some specimens within a week, but was rarely seen in unhatched eggs. Fungus penetration holes in female and cyst walls were observed 3 days after inoculation; penetration through nematode orifices was not seen at that time. More cysts than females were colonized at the earliest sampling dates. Specimens associated with external hyphae exhibited variable internal colonization, ranging from no fungal penetration to extensive mycelial growth.     

Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases

Fungi in the genus Lecanicillium (formerly classified as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

An alternative insect pathogenic strategy in an Aspergillus flavus auxotroph

In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223-232). Here, we report that on 1 % insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.     

Mutualism and asexual reproduction influence recognition genes in a fungal symbiont

Mutualism between microbes and insects is common and alignment of the reproductive interests of microbial symbionts with this lifestyle typically involves clonal reproduction and vertical transmission by insect partners. Here the Amylostereum fungus–Sirex woodwasp mutualism was used to consider whether their prolonged association and predominance of asexuality have affected the mating system of the fungal partner. Nucleotide information for the pheromone receptor gene rab1, as well as the translation elongation factor 1α gene and ribosomal RNA internal transcribed spacer region were utilized. The identification of rab1 alleles in Amylostereum chailletii and Amylostereum areolatum populations revealed that this gene is more polymorphic than the other two regions, although the diversity of all three regions was lower than what has been observed in free-living Agaricomycetes. Our data suggest that suppressed recombination might be implicated in the diversification of rab1, while no evidence of balancing selection was detected. We also detected positive selection at only two codons, suggesting that purifying selection is important for the evolution of rab1. The symbiotic relationship with their insect partners has therefore influenced the diversity of this gene and influenced the manner in which selection drives and maintains this diversity in A. areolatum and A. chailletii.     

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed. The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus. Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex¹. Here we present a protocol that we have developed, based on the artificial infestation technique by Houston², which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts. The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera)

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at −80 °C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.     

Boomeranging in structural defense: Phytophagous insect uses cycad trichomes to defend against entomophagy

Plant defensive behaviors that resist arthropod herbivory include trichome-mediated defenses, and variation in plant trichome morphology and abundance provides examples of the mechanistic complexities of insect-plant interactions. Trichomes were removed from Cycas revoluta cataphylls on the island of Guam to reveal Aulacaspis yasumatsui scale infestation, and predation of the newly exposed insects by pre-existing Rhyzobius lophanthae beetles commenced within one day. The quotient of predated/total scale insects was 0.5 by day 4 and stabilized at that found on adjacent glabrous leaves in about one week. The trichome phenotype covering the C. revoluta cataphyll complex offers the invasive A. yasumatsui armored scale effectual enemy-free space in this system. This pest and predator share no known evolutionary history with C. revoluta, therefore, the adaptive significance of this plant behavior in natural habitat is not yet known.     

Infection and colonization of tissues of the aphid Myzus persicae and cassava mealybug Phenacoccus manihoti by the fungus Beauveria bassiana

Histopathogenesis of living insects of Myzus persicae Sulzer (Hemiptera: Aphididae) and Phenacoccus manihoti Matile‐Ferrero (Hemiptera: Pseudococcidae) by Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was monitored from penetration through insect death. Important events in aphids included fungal penetration of the integument of the less-resistant leg intersegmental membrane and invasion of natural openings, formation of hyphal bodies in live aphids by three days post-inoculation (PI), and extensive hyphal colonization of the two leg segments closest to the insect body at death of the aphids. Confocal microscopy of green fluorescent protein-labeled B. bassiana in live mealybugs indicated the fungus penetrated the host through the legs and mouthparts. The fungus was scarce in live mealybugs at 1–5 days PI, formed hyphal bodies by six days PI, and growth was limited to parts of dead hosts at 6–7 days PI. In dead mealybugs, hyphal bodies were near solid tissue. Blastospores were in the hemolymph.     

Beauveria bassiana: endophytic colonization and plant disease control

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.     

Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum

Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l⁻¹ can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.