Nonphytotoxic copper oxide nanoparticles are powerful“nanoweapons” that trigger resistance in tobacco against the soilborne fungal pathogen Phytophthora nicotianae

CHEN Juan-ni ,WU Lin-tong ,SONG Kun ,ZHU Yun-song ,DING Wei

1 Laboratory of Natural Product Pesticides,College of Plant Protection,Southwest University,Chongqing 400715,P.R.China

2 Jingxi Marketing Department of Basie of Guangxi Tobacco Company,Baise 533899,P.R.China

Abstract Investigations into the potential application of nanoparticles acting as nanofungicides in sustainable agriculture are rapidly expanding due to the high antimicrobial properties of these compounds,which do not risk inducing pathogen resistance to fungicides. A detailed understanding of the impact of copper oxide nanoparticles (CuO NPs) on soil-borne phytopathogenic fungi is yet to be obtained. This study aimed to explore the in vitro antifungal activity and control efficacy of CuO NPs applied via irrigation with respect to tobacco black shank (TBS) disease caused by Phytophthora nicotianae.The results revealed that CuO NPs greatly interfered with the reproductive growth process of this fungus,repressing hyphal growth,spore germination and sporangium production. Additionally,morphological damage,intracellular ROS accumulation and increased SOD enzyme activity in hyphae were the antifungicidal mechanisms of these NPs. In pot experiments,treatment with CuO NPs at 100 mg L-1 significantly suppressed TBS development,compared with the effect on control plants,and the control efficacy reached 33.69% without inducing phytotoxicity. Exposure to CuO NPs significantly activated a series of defense enzymes,and resistance genes in tobacco can further explain the mechanisms by which CuO NPs suppressed fungal infection. The Cu content in both the leaves and roots of P.nicotianae-infested plants increased by 50.03 and 27.25%,respectively,after treatment with 100 mg L-1 CuO NPs,compared with that of healthy plants. In particular,a higher Cu content was observed in infected roots than in leaves. Therefore,this study showed the potential of CuO NPs applied as nanofungicides and as nanoinducers of fungus resistance genes for the management of TBS through inhibition of pathogen infection and stimulation of plant defenses.

Keywords: Nicotiana tabacum L.,tobacco black shank disease,cupper oxide nanoparticles,fungicidal activities,defense response stimulant

1.Introduction

To date,fungal diseases account for approximately 70-80% of crop diseases,significantly reducing agricultural production and even causing disastrous reductions in yields and negative economic impacts in agriculture. It is roughly estimated that more than 8 000 species of phytopathogenic fungi have been identified to date (Fisheret al.2012).Phytophthoranicotianae,a filamentous fungus belonging to the oomycetes class,is one of the most threatening soil-borne fungal pathogens and can attack the roots of tobacco (NicotianatabacumL.) during their whole growing stage,posing a threat to crop production worldwide (Bittner and Mila 2016).Although various alternative control strategies have been applied for protective control,including conventional fungicides and the improvement of cultivation measures(Jiet al.2014;Fanget al.2016),the development of pathogenic fungi resistance and adverse effects caused by environmental exposure to fungicides are emerging threats that have become great challenges to agricultural production. Therefore,under these circumstances,more effective and amicable disease management strategies are urgently needed to meet the criteria for sustainable agriculture.

Significant advances have recently been made in the area of nanomaterials for agricultural applications as novel antimicrobial agents with improved effectiveness because of their unique physical and chemical properties (Gogoset al.2012). The direct fungistatic/fungicidal effects of a multitude of inorganic metal/metal oxide nanoparticles and carbon-based nanomaterials on phytopathogenic,foodborne,and medical fungal pathogens,such as TiO2,ZnO,Ag NPs,graphene oxide (GO) and carbon nanotubes (CNTs),are well documented (Chenet al.2016a,2019;Liuet al.2017;Shenashenet al.2017;Sunet al.2018). Among these nanomaterials,copper oxide nanoparticles (CuO NPs),simple,inexpensive,stable and easily available copper compounds with a high surface area and crystal morphology,are suggested to suppress root bacterial and fungal diseases when applied either the dip method or foliar spray as inhibitors with efficient antimicrobial effects (Meghanaet al.2015;Devipriya and Roopan 2017;Borgattaet al.2018;Elmeret al.2018;Haoet al.2019). Previously,our group proved that CuO NPs possessed strong antibacterial activity against soilborneRalstoniasolanacearum,the cause of bacterial wilt (Chenet al.2019). Elmeret al.(2018) applied CuO and other metal oxide nanoparticles (AlO,FeO,MnO,and ZnO) through foliar spray to effectively controlFusarium oxysporumandVerticilliumdahliaeKlebahn infections on tomato and eggplant plants grown in disease-infested soil,and they were fungitoxic than the corresponding reagents applied in bulk (Elmer and White 2016;Malandrakiset al.2019). Another study demonstrated that Cu NPs and CuO NPs showed strong fungitoxic activity against several foliar and soil-borne plant fungal pathogens,such asPodosphaerapannosaandBotrytiscinerea,invitroand in the detached tests upon foliar application,compared to treatment with TiO2and Fe2O3(Haoet al.2017,2019).Additionally,Giannousiet al.(2013) found that Cu/Cu2O composite NPs exhibited more stable fungistatic activity on late blight of tomatoinvivothan Cu or Cu2O alone(Giannousiet al.2013).

Most importantly,as an essential trace mineral element,copper plays important roles in activating the defense response of plants against diseases,insects,and abiotic stress (Sathiyabama and Manikandan 2018). This outcome is a result of copper involvement in the redox reaction in plants,forming cuprin,which participates in photosynthesis and is an important component of defense enzymes (Sathiyabama and Manikandan 2018;Shanget al.2020). Although Cu nanotoxicity has been reported,low-dose Cu-based nanomaterials profoundly promote the growth and yield of several crops and increase the uptake and translocation of Cu in both greenhouses and field trials (Rastogiet al.2017;Toqeeret al.2020;Wanget al.2020). Notably,the mechanisms of Cu nanomaterials to combat disease as nanopesticides,inducing resistance to pathogens,are related to the upregulation of the expression of plant defense-related genes under pathogen stress,such as inFusariuminfected in watermelon and tomato,leading to more effective translocation through the cuticle,regardless of composition (phosphatevs.oxide) (Borgattaet al.2018;Maet al.2020). However,although manyinvitrostudies have been performed,it is important to remember that nanomaterials invariably exert biological effects depending on the plant species,as well as nanoparticle type,exposure concentration and application process.There have been no systematic studies conducted on the antimitotic properties of CuO NPs against the soil-borne fungal pathogenP.nicotianae. Additionally,it is notable that available studies have applied nanoscale Cu as a foliar sprayinvivo,and information on the management of crop disease by nanoparticlesviasoil application remains largely unclear. In consideration of the considerable the advantages of NPs over traditional fungicides,an allround understanding of how NPs affect pathogens,host plants,and disease control is necessary for efficient application in agriculture (Giannousiet al.2013).

Therefore,the objective of this study was to investigate theinvitrofungitoxicity properties of CuO NPs againstP.nicotianaeand the disease control efficacy upon root irrigation application of CuO NPs based on greenhouse studies. Seed germination and tobacco plant growth were also evaluated in the presence of these nanoparticles.Furthermore,in healthy and infected plants exposed to CuO NPs,the Cu content in plant tissues was determined by inductively coupled plasma-mass spectrometry(ICP-MS),and the defensive enzymatic and molecular responses (the transcriptional levels of defense-related genes) were quantified to validate the ability of these NPs to stimulate tobacco plant disease resistance. The results broaden the horizon to understand the mechanism of CuO NPs against phytopathogens and show promise for the potential application of nanoparticles in an alternative disease management strategy that improves sustainable agricultural applications in the future.

2.Materials and methods

2.1.Nanoparticle characterization

CuO NPs (40 nm,99.00% pure) were purchased from Sigma-Aldrich (Shanghai) Trading Co.,Ltd.(Shanghai,China). The CuO NPs were dispersed in distilled water containing a nonionic surfactant (1 mL L-1) to obtain a series of concentration suspensions. After sonication for 5 min,the particle morphology was observed by transmission electron microscopy (TEM,Hitachi HT7800,Japan). The diameter of CuO NPs ranged from 10 to 100 nm,with some agglomeration (Appendix A).

2.2.Phytophthora nicotianae culture and sporogenesis induction

Phytopathogenic fungusP.nicotianaewas provided by the State Key Laboratory of Crop Stress Biology for Arid Areas,Northwest Agriculture &Forest University.Phytophthoranicotianaewas inversely cultured on oatmeal agar medium (OA) at 28°C in a constant thermostatic incubator in the dark to obtain an inoculum for use in fungitoxicity assays.

Phytophthoranicotianaeconidia were cultured as follows: First,all glass apparatuses and materials used in the experiment were autoclaved at 121°C for 30 min for total sterilization before use.Phytophthoranicotianaeinoculum routinely grown for 4 d was transferred into a Petri dish containing sterilized zoospore-inducting medium consisting of 0.004 mmol L-1MgSO4·7H2O,0.05 mmol L-1KNO3,and 1 mL Fe-EDTA in 1 L ultrapure water. The pH value was adjusted by 2-(n-morpholine)ethylsulfonic acid to 6.0. Then,the inoculum mixture was cultured in an illumination incubator at 26°C with 24 h of constant light until massive sporangium production was observed. After discarding the induction medium,the inoculum was sustained at 4°C for 15 min and then at room temperature for 30 min. Finally,a large number of spores were released from sporangium by gentle blowing with a pipette containing deionized water.Then,the conidial suspension was filtered through three layers of sterile gauze,collected by centrifugation,and counted using a hemocytometer. The spore density was determined in distilled water to be approximately 1×106conidia mL-1.

2.3.In vitro fungitoxicity of CuO NPs against P.nicotianae

The antifungal activities of the CuO NPs were evaluated by measuring the vegetative and reproductive growth of fungi in terms of the inhibition of fungal growth,spore germination and sporangium formation (Chenet al.2020).

Mycelium growth inhibition testsA piece of circularP.nicotianaemycelia cake (approximately 0.6 cm) cut from the edge of a 5-d-old fungal colony was inoculated on oatmeal (OA) agar medium treated with different concentrations (0,6.25,12.5,25,50,and 100 mg L-1) of CuO NPs. All the culture plates were kept in a thermotank at 28°C for 72 h in the dark,and mycelial colony diameters were measured. The mycelial growth inhibitory rate was calculated according to the following formula:

where C indicates the mean diameter of colony in control groups and T indicates the mean diameter of colony in different concentrations of CuO NPs-treated groups.

Prevention of sporangium formationBriefly,CuO NPs were added to spore-inducting medium to reach the tested concentration in the experiment described above,shaken and sterilized,after which 3 mL of the suspension was poured into a culture dish with an inner diameter of 8.0 cm. As a control sample,the same volume of sterile water was used in the experiment. Then,three fungal plugs of the same size grown for 15 d on OA medium were inoculated in each plate. All the untreated and treated plates were sealed with parafilm,followed by exposure to light incubation at 26°C for 24 h to stimulate the formation of sporangium. Finally,the number of sporangia was counted,and images were taken using a Leica DMI 300B microscope (Leica,Germany).

Spore germination assaySimilarly,10 μL of different concentrations of CuO NPs and the same volume ofP.nicotianaespore suspensions (1×106conidia mL-1)were coinocculated on hollow glass slides,which had been cleaned with ethanol. The glass slides were placed in the culture dish and covered with soaked filter paper on the bottom to maintain a relative humidity of 85-95% during the germination process. Following 4 h of incubation at 26°C under completely dark conditions,the spore germination rate was monitored under an optical microscope (Leica,Germany). Typically,the emergence of a germ tube (half of the spore length) is the criterion for spore germination.

2.4.Mycelium morphological observation by SEM/EDS and TEM

Phytophthoranicotianaehyphal morphology changes were observed using SEM and TEM technologies as described previously. AnP.nicotianaeblock was inoculated on 50 mg L-1CuO NP-treated agar medium and cultured for 72 h. Samples ofP.nicotianaehyphae were collected from the edge of the plates and immediately fixed in 3% glutaraldehyde in sodium phosphate buffer(100 mmol L-1,pH 7.4) at room temperature for 24 h.After washing with the buffer several times,the samples were postfixed with 1% osmium tetroxide for 2 h at 4°C.Next,the hyphae were gradually dehydrated in a graded ethanol series,from 30 to 100%,for 10 min. The fixed samples were dried,and the gold coating sputtering procedure was performed. Afterward,the samples were sent to an FEI model Quanta 200 SEM for examination.

For TEM observations,theP.nicotianaehyphae were dehydrated,embedded in epoxy resin,and cut with an ultramicrotome (RMC) to obtain ultrathin sections.Subsequently,all the sections were poststained with 2% uranyl acetate for 10 min and 2% lead citrate for another 10 min. Samples were mounted on a copper grid and examined on a JEM-200 CX (JEOL) TEM at an accelerating voltage of 100 kV (SIA,Germany).

2.5.Viability assay fungal hyphae

Fungal cell viability was assessed using a fluorescence staining assay,according to a previous study (Chenet al.2014).Phytophthoranicotianaehyphae was mixed with a stock solution of CuO NPs (100 mg L-1) and incubated at 28°C for 4 h. A hypha group without treatment served as the control. The susceptibility of the mycelium exposed to nanoparticles as well as that of normal cells was examined with a fluorescence microscope (DM750,Leica Co.,Germany) followed by diamidine phenyl indole (DAPI)and propidium iodide (PI) staining.

2.6.Determination of intracellular ROS levels and superoxide dismutase (SOD) enzyme activity

Intracellular generation of ROS was measured by performing an assay with nonfluorescent 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA,Invitrogen,USA),an oxidative stress indicator,according to a report by Chenet al.(2014). FreshP.nicotianaemycelium were collected and exposed to CuO NPs at concentrations of 100 mg L-1in centrifuge tubes at 28°C for 4 h. Mycelium supplemented with sterile deionized water was the positive control. Then,10 μmol L-1H2DCFDA was added to each tube,and the tested samples were incubated for 20 min in the dark and then washed.The mycelium was separated and inspected under a fluorescence microscope (DM750,Leica Co.,Germany)at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

For the SOD enzyme assay,0.2 g of hyphae were ground with liquid nitrogen in a mortar,and the homogenate was quickly centrifuged at 12 000×g at 4°C for 20 min. The supernatant was used for SOD enzyme activity measurements,which were obtained with a SOD Activity Assay Kit (BioVision,USA),and the reaction was measured with a microplate reader (Thermo Varioskan Flash,USA) at 505 nm. Three replicates for each treatment were prepared. Additionally,parallel experiments were conducted in which a series of dosages of histidine,an antioxidant,was applied to CuO NPexposed fungal hyphae to inhibit the production of SOD.

2.7.Pathogen inoculation and disease control assay in vivo

Tobacco (Yunyan 87) seeds were firstly sterilized using 75% ethylalcohol for 30 s,and then immersed in 30%H2O2for another 10 min. Then,seeds were planted into a floating seedling system containing commercial matrix(Hunan Xianghui Agriculture Technique Development Co.,Ltd.) and grown until the 4-5-true-leaf stage. Next,10 mL of spore suspension (104spores mL-1) was added to each plant by the soil drench method. A total of 10 mL of CuO NPs at various concentrations were applied by irrigation at 24 h post inoculation. The plants treated with sterile distilled water were the controls. Sixteen uniformly sized plants were included in each treatment. The disease incidence was investigated when wilted leaves and necrotic stalks appeared,i.e.,2 d after CuO NP treatment in our experiment,as defined by Litton based on scoring disease severity on a rating scale of 0-4. Thus,the control efficacy was calculated with reference to the literature.

2.8.Determination of the expression of plant defense-related genes by qPCR

For a gene expression change analysis,0.1 g of fresh root tissues were collected after CuO NP-treated tobacco plants were infected for 1,3,5,and 7 d. Total RNA was extracted with a Sigma-Aldrich Spectrum Plant Total RNA Kit (St.Louis,MO),and the obtained RNA concentration was determined with a Thermo Scientific Microplate Reader (Thermo Varioskan Flash,USA). An iScript?cDNA Synthesis Kit (Bio-Rad,USA) was used for reverse transcription of a 2 μL aliquot of RNA. The primers of the genes are listed in Appendix B. Then,the expression of several tobacco genes relevant to the plant defense response was determined using a One-Step RT-qPCR Kit (Bio-Rad,USA) following the procedure below. PCR was run following a set procedure with predenaturing of the DNA at 95°C for 3 min and denaturing at 90°C for 10 s. Next,40 PCR cycles of 60°C for 30 s and then 95°C for 10 s were run to finish the reaction. DNA was quantitatively measured by measuring the fluorescence at the end of each cycle. Relative expression of the selected genes was analyzed using the 2-ΔΔCTmethod,in whichL25was used as the housekeeping gene.

2.9.Statistical analysis

All the experimental data were presented as the mean of three individual three observations with standard deviation(SD) and statistical significance (P＜0.05 andP＜0.01)among all treatments was analyzed by one-way analysis of variance (one-way ANOVA) followed by Duncan’s test(SPSS 19.0,Chicago,IL,USA).

3.Results

3.1.Inactivation of P.nicotianae mycelial growth

The inhibitory effects of CuO NPs onP.nicotianaegrowth were determined by hyphal colony diameter evaluation using the agar diffusion method. As visually observed in Fig.1-A,compared to the untreated sample,colonies covered the whole plate,and hyphae colony diameters,with a value of 6.23,6.18,and 5.65 cm,were clearly not affected by CuO NP treatment at 6.25-25 mg L-1;however,a significant decrease was observed in samples exposed to CuO NPs at the higher concentrations of 50 and 100 mg L-1. The diameters were reduced to 4.06 and 0 cm with a 38.87 and 100% growth inhibitory rate,respectively. Notably,100 mg L-1CuO NPs showed complete inactivation (100% inhibition ratio) againstP.nicotianaehyphae.

3.2.Suppression of sporangium formation and spore germination

Sporangia,an asexual propagule ofP.nicotianae,can germinate directly through the production of hyphae on host tissue or release abundant zoospores,motile asexual spores,to form vegetative mycelium,and approximately 8-20 zoospores are produced by one sporangium,which sprout rapidly to produce new mycelium (Judelson and Blanco 2005). Visually examining the effects on sporangium formation and spore germination was a useful preliminary method to assess the fungicidal activity of CuO NPs. In contrast to the untreated sample,the CuO NPs significantly and progressively reduced the sporangium number with increasing exposure concentration (6.25-100 mg L-1): The average number of sporangia was 140,44,25,5 and 0,a reduction of 42.61,76.70,86.78,97.04,and 100%,respectively (Fig.2-B),demonstrating that CuO NPs greatly affected the sporogenesis ofP.nicotianae.

Similarly,overall,as the exposure concentration increased,spore germination rates were significantly suppressed upon incubation with CuO NPs (Fig.2-D).Spore germination rates were not impacted statistically after exposure to CuO NPs at 6.25 and 12.5 mg L-1but decreased by 11.59,9.29,87,89.6,and 95.7% at higher treatment dosages,of 25-100 mg L-1,over the control,respectively. An alternative hypothesis suggests that at higher concentrations NPs have increased opportunity to interact with spores,as indicated by the red arrows in Fig.2-D.

3.3.Morphological observation after P.nicotianae hyphae exposure to CuO NPs

In addition,TEM images of fungal cells treated with CuO NPs were taken for the purpose of cellular ultrastructure observation. In contrast with the untreated samples,which possessed dense cytoplasm and an integrated cell envelope,cavitation and cell wall injury were found at multiple sites in the 50 mg L-1CuO NPtreated hyphal cells,indicating thatP.nicotianaehyphae were remarkably damaged (Fig.4-A and C). These changes in hyphal cells corresponded to the SEM results.As shown in Fig.4-E and F,when 100 mg L-1CuO NPs interacted with hyphae,CuO NP aggregates clearly adhered to fungal cell walls outside cells (indicated by red arrows) and even penetrated the cell membrane,probably because of electrostatic interactions and their faster sedimentation rate (Chenet al.2020).

3.4.Phytophthora nicotianae hyphal membrane injury after CuO NP treatment

The CuO NP-induced disinfection ofP.nicotianaemycelium was confirmed by the fluorescent dye staining method,which has been widely applied for evaluating biological cell viability in nanomaterial toxicity studies(Panet al.2013;Chenet al.2014). After staining with propid iumiodide (PI),a type of nuclear staining agent that penetrates the damaged cell membrane and embeds in double-stranded DNA and is usually used in cell apoptosis detectionviathe red fluorescence that is emitted. There was negligible red fluorescence emitted from the hyphae after dying without the presence of CuO NPs,while most of the hyphae exposed to 50 mg L-1CuO NPs were stained light red by the PI dye,indicating that the majority of dead cells (Fig.5) were definitely affected by the destruction of the cell envelope caused by the attachment of CuO NPs,as observed in the aforementioned SEM and TEM images.

3.5.ROS production in response to CuO NP treatment

Accumulation of ROS,namely,superoxide (),hydrogen peroxide (H2O2),and OH·,was measured to identify the intracellular oxidative stress in biological cells induced by metal oxide nanoparticles (Bhabraet al.2009). The nonfluorescent reagent H2DCFDA was employed as an indicator in this assay;when it is oxidized to DCF,green luminescence is emitted.As shown in Fig.5,in comparison to untreatedP.nicotianaemycelium,stronger green fluorescence was emitted from the nanoparticle-hyphal aggregates(Fig.5-B). However,it is interesting to note that the fluorescence intensity was significantly repressed when the antioxidant histidine was added at different concentrations (0.5,1,2,and 3 g L-1) to the CuO NP-exposed hyphae (Fig.5-C). Because histidine contributes to the detoxification of ROS and regulates α-ketoglutarate homeostasis in bacteria in response to oxidative stress (Lemireet al.2010).

Additionally,it is well known that several antioxidative enzymes,such as intracellular superoxide dismutase(SOD),glutathione peroxidase,and thioredoxin peroxidases,are involved in the direct elimination of O2and H2O2to maintain redox homeostasis in cells.As shown in Fig.5-D,SOD activity inP.nicotianaehyphae gradually improved with increasing treatment concentration of CuO NPs;that is,it showed concentration-dependent effects. SOD activity was increased 1.1-,2.7-,3.7-,and 5.3-fold in the presence of CuO NPs applied at various doses,respectively.However,the hyphae were extremely susceptible to the nanoparticles,with nearly all dying,as indicated by electron microscopy observation (Figs.3 and 4). In this regard,it may be suggested that the fungal defense system is stimulated to combat CuO NP-induced oxidative stress (Elmeret al.2018;Kumariet al.2019).

3.6.Suppression of fungal disease in a greenhouse experiment

In the pot experiments,the role of CuO NPs in suppressing TBS diseaseinvivowas determined to verify the results of the laboratory experiment showing the fungitoxicity of CuO NPs. Following inoculation withP.nicotianae,tobacco seedlings were exposed to 6.25-100 mg L-1CuO NPs through irrigation,and the severity of black shank disease was evaluated by the disease index. As expected,inP.nicotianaeinfected plants,there was no change in the disease index with low-dose treatment (6.25-25 mg L-1) and little effect on disease control compared to control plants(Fig.6-B). However,treatment with 50 and 100 mg L-1CuO NPs resulted in significant disease suppression,with control efficiencies of 6.52 and 33.69% at the end of the experiment,respectively. The results from pot experiments were congruent with those of theinvitroassay regarding the toxicity of the nanoparticles againstP.nicotianae. There were negligible negative effects on the pathogens exposed to low concentrations of CuO NPs,with no effects on the reproductive growth of the pathogens. Representative images of black shank disease suppression after 8 dpi are presented in Fig.6-A.Obviously,the untreated tobacco seedlings developed kraurotic and wilt symptoms,whereas most of the plants remained bright green,although old leaves exhibited chlorotic phenomena after irrigation application. Most importantly,there were no significant differences in seed germination and morphological indices among treatments after exposure to CuO NPs for 30 d under greenhouse conditions (Appendices C and D),showing the advantage of being non-toxic.

3.7.Plant defense enzyme activity

The activity of several antioxidant enzymes in tobacco roots was investigated to evaluate the plant response to effective CuO NPs treatments (50 and 100 mg L-1)in a greenhouse study. As shown in Fig.6-D,in the absence of NP treatments,P.nicotianaeinoculation significantly increased polyphenol oxidase (PPO) and chalcone isomerase (CHI) activity in roots by 0.62-and 1.23-fold,respectively,but phenylalanine ammonialyase (PAL) and peroxidase (POD) activities remained unchanged or decreased,indicating that the fungal pathogen alone moderately activated plant immunity.Interestingly,the irrigation addition of CuO NPs intensified the tobacco plant resistance response toP.nicotianae.In infected plants,PPO,POD,PAL,and CHI activities in roots exposed to different concentrations of CuO NPs were significantly upregulated relative to the diseased controls,specifically increasing by 91.6,37,33.8,and 65.1% in the 100 mg L-1CuO NP treatment,respectively,probably due to the use of a large dose of NPs. Under healthy conditions (in the absence of the pathogen),plant defense enzyme levels were all statistically unaffected by the treatments,outcomes probably associated with the application concentration of the NPs,similar to soybean treatment with Cu-based NMs (Maet al.2020). Because CuO NPs show promise as nano-enabling plant defense stimuli to promote crop health,especially in the presence of pathogens (Maet al.2020;Sarkaret al.2020).

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3.8.Defense response gene expression

To validate the abovementioned results showing that CuO NPs can induce disease resistance in tobacco,the expression of five plant defense-related genes in roots was investigated. As shown in Fig.7,it is important to note that the transcription levels of the tested genes in tobacco roots were dynamic and varied with the processing time. Roots exposure to CuO NPs at 50 mg L-1did not affect the defensive genes expression in infected plants at the early stage (1 and 3 days after treatment (DAT),but the expression was enhanced at 7 DAT across all time points. However,irrigation treatedP.nicotianae-infested plants with CuO NPs at 100 mg L-1prominently improved expression of genes (hrs203J,NmIMSP,PR1b,P450-1,P450-2) in roots as compared to untreated group. The accumulation of different transcripts were reached maximum on 5 DAT,increased by 3.64-,4.97-,3.43-,7.71-,and 50.32-folds,respectively,and then decreased at 7 DAT,exceptPR1gene. That is because the mitigatory disease burden on treated plants caused reduced expression levels of defensive genes at the end of the experiment.

3.9.Cu content in tobacco leaves and roots

Given that the delivery of Cu to the plant is critical to the observed disease suppression,the Cu content in leaves and roots ofP.nicotianae-infected and heathy tobacco seedlings exposed to CuO NPs was measured. As shown in Fig.7-F,significantly greater Cu content was found inP.nicotianae-infected plants after exposure to CuO NPs. In particular,the root and leaf Cu concentrations were increased by 50.03 and 27.25%,respectively,after 100 mg L-1CuO NP treatment compared to the infected control,revealing that higher levels of Cu accumulated in diseased plants. Interestingly,there was a clear trend showed that application of CuO NPs resulted in significantly increased Cu content in diseased roots,which was 1.95-and 1.85-fold greater than that in leaves,and a similar result was observed in healthy samples when CuO NPs were applied by irrigation (Fig.7-F). However,no significant difference in the Cu levels in either roots or leaves was evident upon NP exposure,except for the Cu level in roots exposed to 100 mg L-1CuO NPs.

4.Discussion

In our study,the antifungal activities of CuO NPs againstP.nicotianaewas higher than againstAspergillusniger,Aspergillusflavus,Fusariumoxysporum,Gloeophyllum trabeumandT.versicolorat the same dosage as previously reported (Weitzet al.2015;Terziet al.2016;Devipriya and Roopan 2017;Herrnida-Monteroet al.2019),probably because of the species-specific effects of the nanoparticles.However,some reports revealed that CuO NPs significantly suppressed fungal mycelial growth only at a 50 mg L-1(Haoet al.2017,2019). Similar strong fungicidal activity of Cu-based NPs against several crop pathogenic fungiinvitrowas observed (Kanhedet al.2014). Theinvitroandin plantasporicidal properties of engineering nanoparticles on various plant fungal species have been reported (Saviet al.2013;Chenet al.2016b). Noticeably,toxicological studies on many pathogens showed better toxicity activity of CuO NPs in comparison to commercial or bulk CuO,although the efficiency greatly depended on different treatment concentrations and conditions of the experiments(Heinlaanet al.2008). This NP superiority was also proven for other metal oxide nanoparticles,such as nano-MgO,ZnO,TiO2,and has been ascribed to their dispersibility,size,and physiochemical properties (Heinlaanet al.2008;Caiet al.2018).

CuO NP toxicity may be derived from a common interaction mechanism,i.e.,the attachment of nanoparticles to fungal cells,which forms unique nanoparticle-spore aggregates. This has been proven in a large number of literatures (Wanget al.2006;Donget al.2013;Hanset al.2013;Meghanaet al.2015;Chenet al.2016b). Mishraet al.(2014) reported that Ag NPs accounted for complete inhibition of conidial germination ofB.sorokiniana. In Fig.3,a large amount of CuO NPs and their aggregates were observed aroundP.nicotianaeunder an optical microscope,especially at higher treatment concentrations (indicated by red arrows). It is likely that nanoparticles form complexes with galactomannans and glycoproteins on the surface of biological cells that inhibit fungal conidiogenesis (Judelson and Blanco 2005). Glycoproteins with adhesive properties fixed on the fungal cell call may play crucial roles in the process of nanoparticle-fungal cell interfacial interactions(Bamfordet al.2015). Importantly,direct contact between the pathogenic fungus and nanoparticles is necessary to induce fatal damage to the conidium body,where the nanoparticles surround and cover the conidia.

A large number of studies on the antimicrobial activity of nanoparticles have shown that cell morphological damage may be critical for agricultural fungal and bacterial cell death (Arciniegas-Grijalbaet al.2017;Caiet al.2018;Chenet al.2019). Although uniform thickening of the cell wall was observed with high-resolution optical microscopy (HROM) upon nanoparticle exposure,dramatic liquefaction of the cytoplasmic contents was also evident and caused reduced electron density (Arciniegas-Grijalbaet al.2017). However,in this experiment,some nanoparticles and aggregates were observed in the cells,possibly because of the nanosize diameter of the pores in the cell walls (2-3 nm) of the hyphae and sporangia(2-4 nm) (Money 1990). Only a single small particle and better dispersibility can enter the spore by penetrating the cell wall. Therefore,it is difficult to distinguish CuO NPs in cells by TEM. We speculate that the adsorbed CuO NPs increased the membrane permeability of fungi significantlyviathe same interaction mode with which they engage bacteria (Meghanaet al.2015;Chenet al.2019). It is likely that metal NPs function by disrupting multiple pathways,including by damaging the bacterial cell wall and inducing intracellular oxidative stress and by interacting with small molecules,some of which have verified in subsequent studies.

In addition,some other stress enzymes are primarily involved in the stress response (Kumariet al.2019). It has been proposed thatP.nicotianaeexposed to higher concentrations of nanoparticles shows increased in ROS generation,thus leading to more severe injury to a range of cellular biological pathways in fungal cells,including cell membrane lipid peroxidation and protein and nucleic acid oxidative damage (Leunget al.2014;Herrnida-Monteroet al.2019). Our results were in agreement with previously obtained results,in which the expression of stress response genes inSclerotiniahomoeocarpawere promoted by Ag NPs (Liet al.2017). Other enzymes related to the cell wall biosynthesis process,such asN-acetylglucosamine andβ-1,3-D-glucan synthase (FKs1p),were inactive,even though the mechanism may be due to the action of dissolved metal iron leached from the nanoparticles.

Similar results were observed with tomato(Lycopersiconesculentum) plants againstPhytophthora infestansupon CuO NP and other copper-based nanomaterials (Cu2O NPs and Cu/Cu2O composite) foliar application under field conditions,efficiently suppressingP.infestansinfection on tomato leaves (Giannousiet al.2013). Moreover,Cu NPs and CuO NPs significantly repressed fusarium wilt,verticillium wilt and bacterial wilt of vegetable species in both greenhouse and field studies after foliar application (Elmer and White 2016;Borgattaet al.2018;Chenet al.2019),while cupperbased nanocomposites exhibited strong antimicrobial properties against phytopathgens (Antonoglouet al.2018;Younget al.2018),which can be considered effective and sustainable antifungal agents for plant bacterial and fungal disease management. However,there are very few studies on root irrigation with nanoparticles. Most notably,sinceP.nicotianaeis present in the roots or rhizosphere soil and the nanoparticles are applied as soil treatments in this study,CuO NPs and pathogens showed certain direct contact (hyphae or spores) to exert superior antifungal action. Therefore,from this point of view,root irrigation is possibly more conducive than foliar application to fighting the pathogen itself. In addition,it is important to note that particle morphology,application method and treatment time were considered crucial factors for nanoparticleinvitrotoxicity evaluation andinvivodisease treatment(Ingle and Rai 2017;Borgattaet al.2018).

Plants employ comprehensive strategies to enhance their tolerance under abiotic and biotic stress,including structural and physiobiochemical changes. Our findings found that antioxidant enzyme activities in plant tissue were improved after treatment with CuO NPs,probably to eliminate excessively accumulated ROS as a result of the biochemical response triggered by pathogen infection(Shenet al.2020). Similarly,upregulation of defenserelated antioxidant genes were observed inFusariuminfested plant roots treated with Cu-based nanomaterials(NMs) (Zhaoet al.2017;Elmeret al.2018;Maet al.2019). Another investigation proved that root PAL and POD contents were generally activated by the same copper nanomaterials and thus increased the induced the tolerance of tomato,lentil and finger millet to fungal pathogens (Sathiyabama and Manikandan 2018;Shenet al.2020). In addition to aforementioned crops grown in soil,in hydroponically cultivated plants,the levels of the enzymatic and nonenzymatic metabolites essential to the plant defense system were stimulated by Cu NPs and thus improved the ability of these plants to fightC.michiganensis(Cumplido-Najeraet al.2019). The resistance signaling pathways activated by nanomaterials is also evident when crops are infected with bacteria(Imadaet al.2016).

However,there were some contradictory findings showing that foliar application of CuO NPs and Si NPs reduced the disease response by reducing shoot and root POD and PPO activity or decreasing the expression of genes related to plant defense,ultimately enhancing the innate crop disease response (Buchmanet al.2019;Maet al.2020). These differences may be due to the plant species,growth stage,nanoparticle concentration,morphology,composition,and,most importantly,application method. In contrast to foliage treatment in most studies,in this study,tobacco roots directly contacted CuO NPs and absorbed copper elements,either in the form of dissolved copper ions or as intact nanoparticles. It stands to reason that applying CuO NPs to roots increased plant sensitivity to Cu stress and Cu availability compared to foliar spraying since root exudates,such as low-molecular-weight organic acids,are able to inhibit the aggregation and facilitate the dissolution of CuO NPs (Penget al.2019),thereby facilitating the induction of plant immunity (elevation of antioxidant enzymes) in tobacco to mitigate the ROS generated by pathogen infection.

In this study,the application of CuO NPs triggered the tobacco immune system by reinforcing the defense mechanism through the modulation of these resistanceassociated genes. The improved transcription of PR protein-1,hrs203JandNmIMSPsuggested that CuO NPs activated the salicylic acid (SA) defense reaction or stimulated the plant hypersensitive response in response to pathogens,inducing the reduction in black shoot disease incidence in the pot experiments (Fig.6-A)(Silvaet al.2013). P450s are multifunctional oxidases that widely exist in plants and can participate in the synthesis of various disease-resistant substances in plants through a variety of metabolic pathways,such as toxins,bactericides and stress signals,to activate plant disease resistance (Glawischnig 2006;Kooet al.2011).The transcript levels of genes were changed,presenting differences on different days in this experiment since gene expression in plants depends on the treatment time,the life-cycle stage of the plant and the infection time of pathogens (Maet al.2019). These positive defense responses make the application of nanomaterials an effective strategy for plant disease control.

In addition,we found that Cu mainly accumulated in the roots and was delivered to leaves. Cu availability in plant roots was considered to be a strong driver of defensive reactions against fungal infection (Shanget al.2020). It is reasonable to speculate that the high Cu levels in infected tobacco may be accountable for the elevated activity of antioxidant enzymes. It has been proven that the application of CuO NPs activates the plant depensation response against pathogen interference,which means that the root uptake and transportation capacity of nutritive elements were extremely high afterP.nicotianaeattacks tobacco plants,thereby resulting in an enhanced Cu content in tissues even at a low dose of copper supplementation. Another reason for this outcome involves the great ability of CuO NPs to release copper ions,especiallyviasoil supplementation,as a result of a series of plant root exudates produced by pathogen infection,which may advantageously dissolve nanoparticles and further largely enhance their adsorption by plants (Penget al.2019). Copper is a micronutrient that serves as an essential factor in most aerobic plants and is an indispensable element in a variety of Cu proteins,which participate in photosynthetic electron transport and host defense responses (Cohuet al.2009;Chmielowskaet al.2010). Plant disease suppression and plant growth promotion were associated with foliar or soil treatment of CuO NPs as nanofertilizers through modulation of nutritional and phytohormone status in plants (Maet al.2020;Perezet al.2020;Shanget al.2020). However,in the case of healthy plants,the Cu content in all of the tissues of CuO NP-treated plants remained statistically unchanged compared to that of the untreated samples.

To our knowledge,this is the first report on the mechanism of CuO NPs suppressP.nicotianaeinfection in tobacco. As indicated in Fig.8,in this study,CuO NPs could be applied as nanoelicitor through root irrigation and an effective fungicide against the fungal pathogenP.nicotianae. The information is of importance for understanding the beneficial use of CuO NPs in sustainable agricultural. Further studies are required to precise visualization of the NPs’ location within the plant tissues and evaluate the disease suppression of CuO NPs under field conditions and their fate in the environment.

5.Conclusion

In summary,this study aimed to first expound the properties of CuO NPs as nano-antifungicides and stimulants of plant resistance responses againstP.nicotianae,the causative agent of blank shank disease in tobacco. Theinvitrodose-responsive fungicidal activity induced by CuO NPs againstP.nicotianaewas investigated,as measured by fungal reproductive growth,which involves mycelial growth,spore germination and sporangium formation. Morphological injury observed by SEM and TEM indicated an additional toxicity-inducing mechanism,which was attributed to the direct interaction between nanoparticles and fungi. Additionally,reduced incidence of TBS after irrigation application of CuO NPs,without causing adverse effects,was observed in a greenhouse study. In addition,an increase in Cu content was observed in both healthy andP.nicotianaeinfected plants compared to untreated plants,which was attributed to the NP-induced enhanced enzyme activity and expression of defense response genes in tobacco.These systematic investigations provid the insight into the environmental behaviors of CuO NPs on agricultural soil-borne fungi and the insight for the development of efficient,alternative,nano-enabled fungicides against soilborne plant pathogenic fungi.

Acknowledgements

The authors acknowledge the financial support by the National Natural Science Foundation of China (32001934)and the Key Science and Technology Project of Sichuan Tobacco Company,China (SCYC202114).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm