Subchronic toxicity of dietary sulfamethazine and nanoplastics in marine medaka (Oryzias melastigma): Insights from the gut microbiota and intestinal oxidative status.

Antibiotics and nanoplastics are two prevalent pollutants in oceans, posing a great threat to marine ecosystems. As antibiotics and nanoplastics are highly bioconcentrated in lower trophic levels, evaluating their impacts on marine organisms via dietary exposure route is of great importance. In this study, the individual and joint effects of dietborne sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) in marine medaka (Oryzias melastigma) were investigated. After 30 days of dietary exposure, 4.62 mg/g SMZ decreased the Chao1 index (60.86% for females and 26.85% for males) and the Shannon index (68.95% for females and 65.05% for males) and significantly altered the structure of gut microbial communities in both sexes. The female fish exposed to 4.62 mg/g SMZ exhibited higher intestinal sod (43.5%), cat (38.5%) and gpx (39.6%) transcripts, indicating oxidative stress in the gut. PS alone at 3.45 mg/g slightly altered the composition of the gut microbiota. Interestingly, the mixture of SMZ and PS caused more modest effects on the gut microbiota and intestinal antioxidant physiology than the SMZ alone, suggesting that the presence of PS might alleviate the intestinal toxicity of SMZ in a scenario of dietary co-exposure. This study helps better understand the risk of antibiotics and nanoplastics to marine ecosystems.

Long-Term Effects of Polyvinyl Chloride Microplastics on Anaerobic Granular Sludge for Recovering Methane from Wastewater.

Polyvinyl chloride microplastics (PVC-MPs) are emerging contaminants affecting biological wastewater treatment processes. However, most of the previous studies mainly focused on their short-term impacts on floc sludge, with little work being conducted to explore their potential effects on more complex anaerobic granular sludge (AGS), which has been widely used for high-strength organic wastewater treatment. In this paper, the long-term effects of PVC-MPs on AGS were investigated via continuous feeding tests that are representative of real wastewater treatment processes. The results of a continuous 264 days test showed that the prolonged exposure of PVC-MPs at 15-150 MPs·L-1 significantly (p = 7.86 × 10-37, 3.44 × 10-43, and 5.29 × 10-46) inhibited the COD removal efficiency of AGS by 13.2%-35.5%, accompanied by a 11.0%-32.3% decreased production of methane and 40.3%-272.7% increased accumulation of short-chain fatty acids (SCFAs). In addition, the PVC-MPs exposure suppressed the secretion of extracellular polymeric substances (EPS), causing AGS and the inside microorganisms to lose the protection of EPS, thereby resulting in granule breakage and decreased cells viability. Aligning with the deteriorated performance, the long-term exposure of PVC-MPs reduced the total microbial populations and the relative abundances of key methanogens and acidogens. A toxicity mechanism assessment revealed that the negative impacts induced by PVC-MPs are mainly attributed to the toxic leachate and excess oxidative stress.

Surface functional groups on nanoplastics delay the recovery of gut microbiota after combined exposure to sulfamethazine in marine medaka (Oryzias melastigma).

Nanoplastics can interact with antibiotics, altering their bioavailability and the ensuing toxicity in marine organisms. It is reported that plain polystyrene (PS) nanoplastics decrease the bioavailability and adverse effects of sulfamethazine (SMZ) on the gut microbiota in Oryzias melastigma. However, the influence of surface functional groups on the combined effects with SMZ remains largely unknown. In this study, adult O. melastigma were fed diet amended with 4.62 mg/g SMZ and 3.65 mg/g nanoplastics (i.e., plain PS, PS-COOH and PS-NH2) for 30 days (F0-E), followed by a depuration period of 21 days (F0-D). In addition, the eggs produced on the last day of exposure were cultured under standard protocols without further exposure for 2 months (F1 fish). The results showed that the alpha diversity or the bacterial community of gut microbiota did not differ among the SMZ + PS, SMZ + PS-COOH, and SMZ + PS-NH2 groups in the F0-E and F1 fish. Interestingly, during the depuration, a clear recovery of gut microbiota (e.g., increases in the alpha diversity, beneficial bacteria abundances and network complexity) was found in the SMZ + PS group, but not for the SMZ + PS-COOH and SMZ + PS-NH2 groups, indicating that PS-COOH and PS-NH2 could prolong the toxic effect of SMZ and hinder the recovery of gut microbiota. Compared to plain PS, lower egestion rates of PS-COOH and PS-NH2 were observed in O. melastigma. In addition, under the simulated fish digest conditions, the SMZ-loaded PS-NH2 was found to desorb more SMZ than the loaded PS and PS-COOH. These results suggested that the surface -COOH and -NH2 groups on PS could influence their egestion efficiency and the adsorption/desorption behavior with SMZ, resulting in a long-lasting SMZ stress in the gut during the depuration phase. Our findings highlight the complexity of the carrier effect and ecological risk of surface-charged nanoplastics and the interactions between nanoplastics and antibiotics in natural environments.

Polylactic acid (PLA), polyethylene terephthalate (PET), and polystyrene (PS) microplastics differently affect the gut microbiota of marine medaka (Oryzias melastigma) after individual and combined exposure with sulfamethazine.

Microplastics and the antibiotic sulfamethazine (SMZ) are two prevalent pollutants in regions with high human activity, particularly in coastal marine environments. In this study, the individual and joint effects of microplastics (i.e., the bio-based microplastics polylactic acid (PLA), the petroleum-based microplastics polyethylene terephthalate (PET), and the petroleum-based microplastics polystyrene (PS) at 0.5 and 5 mg/g) and sulfamethazine (SMZ, at 5 mg/g) on the gut microbiota of marine medaka (Oryzias melastigma) via dietary route were investigated. For the individual microplastics exposure, two petroleum-based microplastics PET and PS significantly decreased the alpha diversity and the complexity of co-occurrence networks of gut microbiota. Differently, the adverse effects caused by the bio-based microplastic PLA were more modest, suggesting that PLA was less hazardous than PET and PS. For the combined exposure, SMZ alone dramatically impaired the homeostasis of gut microbiota by decreasing the alpha diversity and the complexity of co-occurrence networks, while the presence of PLA or PET alleviated these adverse effects caused by SMZ. Interestingly, such an alleviation effect was not observed in the SMZ + PS groups, suggesting that different types of microplastics might exhibit distinct joint effects with SMZ. Our findings contribute to a better understanding of the ecological risk of different types of microplastics to marine ecosystems, especially in a scenario of combined pollution with antibiotics.

Incomplete recovery of gut microbiota in marine medaka (Oryzias melastigma) during the depuration phase, after exposure to sulfamethazine/nanoplastics.

The persistence of antibiotics and nanoplastics in aquatic environment poses a great threat to aquatic organisms. In our previous study, significant decreases of bacterial richness and changes of bacterial communities in the Oryzias melastigma gut after sulfamethazine (SMZ) and polystyrene nanoplastics (PS) exposure were observed. Here, the O. melastigma dietary exposed to SMZ (0.5 mg/g, LSMZ; 5 mg/g, HSMZ), PS (5 mg/g, PS) or PS + HSMZ were depurated for 21 days to assess the extent of which these effects were reversible. Our results revealed that most diversity indexes of bacterial microbiota in the O. melastigma gut from the treatment groups were insignificantly different from the control, suggesting a large recovery of bacterial richness. Although the sequence abundances of a few genera remained significantly changed, the proportion of dominant genus was recovered. Exposure to SMZ affected the complexity of the bacterial networks, and the cooperation and exchange events of positively associated bacteria were enhanced during this period. After depuration, increases in the complexity of networks and intense competitions among bacteria were observed, which was beneficial for the robustness of networks. However, the gut bacterial microbiota was less stable, and several functional pathways were dysregulated, relative to the control. In addition, higher occurrence of pathogenic bacteria was found in the PS + HSMZ group relative to the signal pollutant group after depuration, indicating a greater hazard for the mixture of PS and SMZ. Taken together, this study contributes to a better understanding of the recovery of bacterial microbiota in fish gut after individual and combined exposure to nanoplastics and antibiotics.

Understanding and mitigating the distinctive stresses induced by diverse microplastics on anaerobic hydrogen-producing granular sludge.

This work comparatively studied the different stress responses of anaerobic hydrogen-producing granular sludge (HPG) to several typical MPs in wastewater, i.e., polyethylene (PE), polyethylene terephthalate (PET), and polyvinyl chloride (PVC) MPs. A new approach to mitigating the inhibition caused by MPs based on biochar was then proposed. The results displayed that microbe in HPG had diverse tolerances to PE-MPs, PET-MPs and PVC-MPs, with the hydrogen production downgraded to 82.0 ± 3.2 %, 72.3 ± 2.5 % and 66.6 ± 2.3 % (p < 0.05) of control respectively, due to the distinct leachates toxicities and oxidative stress level induced by different MPs. The discrepant mitigation reflected in the hydrogen yields of biochar-based HPGs raised back to 88.7 ± 1.4 %, 85.3 ± 3.8 % and 88.5 ± 3.5 % of control. The MPs induced disintegrated granule morphology, fragile microbial viability and impaired defensive function of extracellular polymeric substances were restored by biochar. The effective mitigation was revealed to be due to the strong adsorption of MPs by biochar, reducing direct contact between microbes and MPs. Biochar addition also enhanced protection for HPG by increasing EPS secretion and weakened the oxidative damage to anaerobes induced by MPs. Biochar manifested the disparate adsorption properties of three MPs. The most superior mitigation in HPG contaminated by PVC-MPs was attributed to the strongest affinity of biochar to PVC-MPs and effective alleviation of PVC leachates toxicity.

Evaluating the role of biochar in mitigating the inhibition of polyethylene nanoplastics on anaerobic granular sludge.

The extensive application of anaerobic granular sludge (AGS) to wastewater treatment for methane recovery has drawn considerable attention to the system performances affected by the presence of emerging contaminants in wastewater such as nanoplastics. However, effective strategies on how to mitigate the inhibition caused by nanoplastics remained unavailable. In this study, a novel strategy using biochar to mitigate the inhibition on the AGS performances caused by polyethylene nanoplastics (PE-NPs) was proposed and the corresponding mitigating mechanisms involved were explored. The PE-NPs solely decreased the level of methane recovery of AGS to 71.3 ± 2.7% of control, which was subsequently increased to 85.6 ± 0.8% of control with the presences of both biochar and PE-NPs, although biochar solely showed no obvious effect on methane production. The addition of biochar also elevated the granule size of AGS, along with AGS integrity based on the morphological observation. Moreover, the distributions of live cells and functional microbes related to acidification and methanation increased with biochar addition compared to sole PE-NPs exposure. More extracellular polymeric substance (EPS) was secreted when biochar was involved in AGS systems, with more protein being detected to maintain the granule structure of AGS. Evaluation of adsorption tests indicated that biochar possessed stronger affinity for PE-NPs than AGS, thus capturing the PE-NPs that would originally contact AGS and posing less toxicity to microorganisms.

Microbial and physicochemical responses of anaerobic hydrogen-producing granular sludge to polyethylene micro(nano)plastics.

Micro(nano)plastics is an emerging contaminant in wastewater that has showed significant impacts on various biological treatment processes. Nevertheless, the underlying effects of micro(nano)plastics with different concentrations and sizes on the anaerobic hydrogen-producing granular sludge (HPG) were still unclear. This work firstly attempted to illustrate the microbial and physicochemical responses of HPG to a shock load of polyethylene microplastics (PE-MPs) with varied concentrations and sizes. The results revealed that the PE-MPs inhibitory effect on hydrogen production by HPG was both concentration- and size-dependent. Specifically, the increase of PE-MPs concentration and the decline of PE-MPs size to nano-sized plastics (NPs) significantly decreased the hydrogen yield, downgraded to 79.9 ± 2.6% and 63.0 ± 3.9% (p = 0.001, and 0.0002) of control, respectively, at higher MPs concentration and the smaller MPs size (i.e., NPs). The higher PE-MPs concentration and PE-NPs also suppressed extracellular polymeric substances (EPS) generation more severely. The critical bio-processes involved in hydrogen production were disturbed by PE-MPs, with the extent of negative impacts depending on the dosage and size of PE-MPs. These adverse impacts further manifested as granule disintegration and loss of cellular activity. Mechanism analysis highlighted the roles of oxidative stress, leachate released from PE-MPs, interaction between PE-NPs and granules inducing physical crushing of HPG that led to possible direct contact between cells and toxic substances.

Multiple microplastics induced stress on anaerobic granular sludge and an effectively overcoming strategy using hydrochar.

Previous studies mostly focused on the responses of anaerobic granular sludge (AGS) to one kind of microplastics during wastewater treatment. However, a wide variety of microplastics has been detected in wastewater. The multiple microplastics induced stress on AGS and the effectively mitigating strategy still remain unavailable. Herein, this work comprehensively excavated the influences of multiple microplastics (i.e., polyethylene terephthalate (PET), polystyrene (PS), polyethylene (PE) and polypropylene (PP)) coexisting in the wastewater on AGS system from macroscopic to microcosmic aspects. Experimental results illustrated that microplastics decreased AGS granule size, increased cell inactivation and caused deteriorative methane recovery from wastewater. As such, this study then put great emphasis on proposing a mitigating strategy using hydrochar and disclosing the role of hydrochar in overcoming the stress induced by coexisting microplastics to AGS system. Physiological characterization and microbial community analysis demonstrated that hydrochar effectively mitigated the reductions in methane production by 50.6% and cell viability by 68.8% of microplastics-bearing AGS and reduced the toxicity of microplastics to microbial community in the AGS. Mechanisms investigation by fluorescence tagging and excitation emission matrix fluorescence spectroscopy with fluorescence regional integration (EEM-FRI) analysis revealed that hydrochar adsorbed/accumulated microplastics and enhanced microplastics-bearing AGS to secrete extracellular polymeric substance (EPS) with more humic acid generation, thus reducing the direct contact between microplastics and AGS. In addition, hydrochar weakened the AGS intracellular oxidative stress induced by microplastics, thereby completely eliminating the inhibition of microplastics on acidification efficiency of AGS, and partially mitigating the suppression on methanation.

Different sizes of polystyrene microplastics induced distinct microbial responses of anaerobic granular sludge.

Recent investigations confirmed the inhibitory effect of microplastics with single sizes on the anaerobic granular sludge (AGS) wastewater treatment system. However, the differences of toxicity from different sizes of microplastics toward AGS and their underlying mechanism are still unclear. In this work, the responds of AGS exposed to different particle sizes of polystyrene microplastics (PS-MPs) were reported. The results showed that the increasing particle sizes (from 0.5 µm to 150 µm) of PS-MPs induced a gradually increasing and distinct inhibitory (from 6.7% to 16.2%) effect on the cumulative methane production by AGS, accompanied by the similar decreasing organic carbon degradation trends. Correspondingly, the integrity and the cell viability of the AGS granules were damaged and the populations of the key acidogens and methanogens were reduced when exposed to PS-MPs, which was particularly evident in the reactors affected by the larger micron-sized PS-MPs. The zeta potential and contact angle indicated that the larger-sized PS-MPs had the stronger dispersive properties and affinity for AGS, causing the higher oxidative stress and leachates toxicity. Further investigation revealed that the tolerance of AGS to PS-MPs toxicity also exhibited size-dependent trend. Larger particles (e.g., 150 µm) of PS-MPs inhibited extracellular polymeric substance (EPS) secretion, while smaller particles (e.g., 0.5 µm) promoted EPS generation with the release of more humic acid, alleviating their toxicity.

Responses of anaerobic hydrogen-producing granules to acute microplastics exposure during biological hydrogen production from wastewater.

Anaerobic hydrogen-producing granule (AHPG) has been successfully applied in hydrogen production from wastewater. While various types of microplastics in large amounts are readily detected in both municipal and industrial wastewaters, however, to date the response of AHPG to multiple coexisting microplastics in wastewater is unknown yet. Herein, this study provided a first insight into the acute exposure-response relationship between multiple coexisting microplastics and the AHPG during biological hydrogen production from wastewater. Fluorescence tagging found that many microplastics accumulated and covered on the surface of the whole granule. Morphology and particle size of microplastics-bearing AHPG were characterized by microscopic observation, showing that the shock load of microplastics in the wastewater at the studied concentrations (40 and 80 mg/L) made the granule loose and even break down with the decreased particle size. The visualization of extracellular polymeric substances (EPS) structure revealed that microplastics decreased EPS production by 8.8-16.7%. Microbial community analysis demonstrated that the acute exposure of microplastics did not drive the change in the microbial community diversity and composition. However, toxic leachates and upgraded oxidative stress induced by microplastics increased cell death up to 14.7% and decreased hydrogen production by 18.7%, when the AHPG exposed to 80 mg/L of microplastics. This work gained a new insight into the response of anaerobic microorganisms to coexisting microplastics in the real environment.

Parental exposure to sulfamethazine and nanoplastics alters the gut microbial communities in the offspring of marine madaka (Oryzias melastigma).

The individual and combined toxicity of antibiotics and nanoplastics in marine organisms has received increasing attention. However, many studies have been mostly focused on the impacts on the directly exposed generation (F0). In this study, intergenerational effects of sulfamethazine (SMZ) and nanoplastic fragments (polystyrene, PS) on the growth and the gut microbiota of marine medaka (Oryzias melastigma) were investigated. The results showed that parental exposure to dietary SMZ (4.62 mg/g) alone and PS (3.45 mg/g) alone for 30 days decreased the body weight (by 13.41% and 34.33%, respectively) and altered the composition of gut microbiota in F1 males (two months after hatching). Interestingly, parental exposure to the mixture of SMZ and PS caused a more modest decrease in the body weight of F1 males than the PS alone (15.60% vs 34.33%). The hepatic igf1 level and the relative abundance of the host energy metabolism related phylum Bacteroidetes for the SMZ + PS group were significantly higher than those for the PS group (igf1, increased by 97.1%; Bacteroidetes, 2.876% vs 0.375%), suggesting that the parentally derived mixture of SMZ and PS might influence the first microbial colonization of gut in a different way to the PS alone. This study contributes to a better understanding of the long-term risk of antibiotics and nanoplastics to marine organisms.

Microplastics decrease the toxicity of triphenyl phosphate (TPhP) in the marine medaka (Oryzias melastigma) larvae.

Plastics have been recognized as a serious threat to the environment. Besides their own toxicity, microplastics can interact with other environmental pollutants, acting as carriers and potentially modulating their toxicity. In this study, the toxicity of polystyrene (PS) microplastic fragments (plain PS; carboxylated PS, PS-COOH and aminated PS, PS-NH2) and triphenyl phosphate (TPhP) (an emerging organophosphate flame retardant) at the environmentally relevant concentrations to the marine medaka (Oryzias melastigma) larvae was investigated. Larvae were exposed to 20 µg/L of microplastic fragments or 20 and 100 µg/L of TPhP or a combination of both for 7 days. The results showed that the three microplastics did not affect the larval locomotor activity. For TPhP, the larval moving duration and distance moved were significantly decreased by the TPhP exposure, with a maximum decrease of 43.5% and 59.4% respectively. Exposure to 100 µg/L TPhP respectively down-regulated the expression levels of sine oculis homeobox homologue 3 (six3) and short wavelength-sensitive type 2 (sws2) by 19.1% and 41.7%, suggesting that TPhP might disturb eye development and photoreception and consequently the low locomotor activity in the larvae. Interestingly, during the binary mixture exposure, the presence of PS, PS-COOH or PS-NH2 reversed the low locomotor activity induced by 100 µg/L TPhP to the normal level. Relative to the larvae from the 100 µg/L TPhP group, the movement duration and distance moved were increased by approximately 60% and 100%, respectively, in the larvae from the TPhP + PS, TPhP + PS-COOH and TPhP + PS-NH2 groups. However, the gene expression profiles were distinct among the fish from the TPhP + PS, TPhP + PS-COOH and TPhP + PS-NH2 groups, implying different mechanisms underlying the reversal of the locomotor activity. The findings in this study challenge the general view that microplastics aggravate the toxicity of the adsorbed pollutants, and help better understand the environmental risk of microplastic pollution.

Insights into the microbial response of anaerobic granular sludge during long-term exposure to polyethylene terephthalate microplastics.

The negative effects of ubiquitous microplastics on wastewater treatment have attracted increasing attention. However, the potential impacts of microplastics on anaerobic granular sludge (AGS) remain unknown. To fill this knowledge gap, this paper investigated the response of AGS to the exposure of model microplastics (polyethylene terephthalate (PET-MPs)) and provided insights into the mechanisms involved. The 84 days' long-term exposure experiments demonstrated that PET-MPs, at relatively low level (15 MP L-1) did not affect AGS performance during anaerobic wastewater treatment, while 75-300 MP L-1 of PET-MPs caused the decreases of COD removal efficiency and methane yields by 17.4-30.4% and 17.2-28.4%, accompanied with the 119.4-227.8% increase in short-chain fatty acid (SCFA) accumulation and particle breakage. Extracellular polymeric substances (EPS) analysis showed that dosage-dependent tolerance of AGS to PET-MPs was attributed to the induced EPS producing protection role, but PET-MPs at higher concentrations (75-300 MP L-1) suppressed EPS generation. Correspondingly, microbial community analysis revealed that the populations of key acidogens (e.g., Levilinea sp.) and methanogens (e.g., Methanosaeta sp.) decreased after long-term exposure to PET-MPs. Assessment of the toxicity of PET-MPs revealed that the leached di-n-butyl phthalate (DBP) and the induced reactive oxygen species (ROS) by PET-MPs were causing toxicity towards AGS, confirmed by the increases in cell mortality and lactate dehydrogenase (LDH) release. These results provide novel insights into the ecological risk assessment of microplastics in anaerobic wastewater treatment system.

Polyethylene terephthalate microplastics affect hydrogen production from alkaline anaerobic fermentation of waste activated sludge through altering viability and activity of anaerobic microorganisms.

Alkaline (especially pH 10) anaerobic fermentation of waste activated sludge (WAS) has been reported to be an effective approach for hydrogen production through inhibiting the homoacetogenesis and methanogenesis. However, the potential effect of the widespread microplastics in sludge on the performance of hydrogen production has never been reported. To fill this knowledge gap, the dominant polyethylene terephthalate (PET) microplastics in WAS were selected as the model microplastics to evaluate their influences on hydrogen production during alkaline anaerobic fermentation of WAS as well as the key mechanisms involved. Experimental results demonstrated that hydrogen production from WAS decreased in the presence of PET microplastics (i.e., 10, 30 and 60 particles/g-TS) compared to the control, with the hydrogen yield at 60 particles/g-TS being only 70.7 ±â€¯0.9% of the control. Although the hydrogen consumption (i.e., homoacetogenesis and methanogenesis) was restrained under alkaline (pH 10) condition, PET microplastics inhibited hydrolysis, acidogenesis and acetogenesis in alkaline WAS anaerobic fermentation, leading to the inhibitory effect on hydrogen production. This was further confirmed by the microbial analysis, which clearly showed PET microplastics caused the shift of the microbial community toward the direction against hydrolysis-acidification. Mechanism studies revealed that PET microplastics carried on their negative influence mainly through leaching the toxic di-n-butyl phthalate (DBP). The reactive oxygen species (ROS) and live/dead staining tests indicated that the increased ROS was induced by PET microplastics, causing more cells dead, which further resulted in the decreased production of hydrogen.

Active changes of lignification-related enzymes in pepper response to Glomus intraradices and/or Phytophthora capsici.

The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal colonization rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was completely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45,000, 53,000 and 114,000) were present in Pc-treated roots, while two major bands (53,000 and 114,000) and one minor band (45,000) were present in spectra of Gi+Pc-treated roots, the 45,000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45,000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60,000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.