Various functions of detoxification enzymes against insecticides in Nilaparvata lugens selected by toxicity assays and RNAi methods.

The brown planthopper (BPH), Nilaparvata lugens is a devastating agricultural pest of rice, and they have developed resistance to many pesticides. In this study, we assessed the response of BPH nymphs to nitenpyram, imidacloprid, and etofenprox using contact and dietary bioassays, and investigated the underlying functional diversities of BPH glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450 monooxygenase (P450) against these insecticides. Both contact and ingestion toxicity of nitenpyram to BPH were significantly higher than either imidacloprid or etofenprox. Under the LC50 concentration of each insecticide, they triggered a distinct response for GST, CarE, and P450 activities, and each insecticide induced at least one detoxification enzyme activity. These insecticides almost inhibited the expression of all tested GST, CarE, and P450 genes in contact bioassays but induced the transcriptional levels of these genes in dietary bioassays. Silencing of NlGSTD2 expression had the greatest effect on BPH sensitivity to nitenpyram in contact test and imidacloprid in dietary test. The sensitivities of BPH to insecticide increased the most in the contact test was etofenprox after silencing of NlCE, while the dietary test was nitenpyram. Knockdown of NlCYP408A1 resulted in BPH sensitivities to insecticide increasing the most in the contact test was nitenpyram, while the dietary test was imidacloprid. Taken together, these findings reveal that NlGSTD2, NlCE, and NlCYP408A1 play an indispensable role in the detoxification of the contact and ingestion toxicities of different types of insecticides to BPH, which is of great significance for the development of new strategies for the sucking pest control.

The brown planthopper NlDHRS11 is involved in the detoxification of rice secondary compounds.

BACKGROUND: The brown planthopper (Nilaparvata lugens, BPH) is the most destructive serious pest in rice production. Resistant varieties are effective means to defend against BPH, but the impact of the ingestion of resistant rice on BPH transcriptional regulation is still unclear. Here, we explore the molecular basis of the regulation by BPH feeding on resistant rice. RESULTS: BPH nymphs preferentially selected susceptible rice TN1 at 24 h after release in a choice test. Feeding on resistant rice IR56 under nonselective conditions increased mortality, decreased growth rate, and prolonged the molting time of BPH. Transcriptomic sequencing revealed 38 dysregulated genes, including 31 down-regulated and seven up-regulated genes in BPH feeding on resistant rice for 7 days compared with feeding on susceptible rice TN1. These genes were mainly involved in the pathways of growth and development, metabolism, energy synthesis, and transport. Finally, we showed that the toxicities of rice defensive compounds to BPH were dose-dependent, and silencing of the BPH gene dehydrogenase/reductase SDR family member 11 (NlDHRS11) increased sensibility to the rice secondary compounds ferulic acid and resorcinol. CONCLUSION: The adaption of BPH feeding on resistant rice is orchestrated by dynamically regulating gene expressions, and NlDHRS11 is a gene involved in the detoxification of plant defensive chemicals. The current work provides new insights into the interaction between insects and plants, and will help to develop novel BPH control strategies. © 2023 Society of Chemical Industry.

Characterization of the First Alternavirus Identified in Fusarium avenaceum, the Causal Agent of Potato Dry Rot.

A novel virus with a double-stranded RNA (dsRNA) genome was isolated from Fusarium avenaceum strain GS-WW-224, the causal agent of potato dry rot. The virus has been designated as Fusarium avenaceum alternavirus 1 (FaAV1). Its genome consists of two dsRNA segments, 3538 bp (dsRNA1) and 2477 bp (dsRNA2) in length, encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein (HP), respectively. The virions of FaAV1 are isometric spherical and approximately 30 nm in diameter. Multiple sequence alignments and phylogenetic analyses based on the amino acid sequences of RdRp and HP indicated that FaAV1 appears to be a new member of the proposed family Alternaviridae. No significant differences in colony morphology and spore production were observed between strains GS-WW-224 and GS-WW-224-VF, the latter strain being one in which FaAV1 was eliminated from strain GS-WW-224. Notably, however, the dry weight of mycelial biomass of GS-WW-224 was higher than that of mycelial biomass of GS-WW-224-VF. The depth and the width of lesions on potato tubers caused by GS-WW-224 were significantly greater, relative to GS-WW-224-VF, suggesting that FaAV1 confers hypervirulence to its host, F. avenaceum. Moreover, FaAV1 was successfully transmitted horizontally from GS-WW-224 to ten other species of Fusarium, and purified virions of FaAV1 were capable of transfecting wounded hyphae of the ten species of Fusarium. This is the first report of an alternavirus infecting F. avenaceum and conferring hypervirulence.

Complete genome sequence of a novel mitovirus isolated from the fungus Fusarium equiseti causing potato dry rot.

In this study, a novel mitovirus was isolated from the fungus Fusarium equiseti causing potato dry rot and tentatively designated as "Fusarium equiseti mitovirus 1" (FeMV1). The full-length genome sequence of FeMV1 consists of 2,459 nucleotides with a predicted A + U content of 69.5%. Using the mold mitochondrial genetic code, an open reading frame (ORF) of 725 amino acids (aa) was predicted to encode an RNA-dependent RNA polymerase (RdRp). The RdRp protein contains six conserved motifs, with the highly conserved GDD in motif IV, and the 5'-untranslated region (UTR) and 3'-UTR of FeMV1 have the potential to fold into stem-loop secondary structures and a panhandle structure, both of which are typical characteristics of members of the family Mitoviridae. Results of a BLASTp search showed that the RdRp aa sequence of FeMV1 shared the highest sequence similarity with that of Fusarium poae mitovirus 2 (FpMV2) (76.84% identity, E-value = 0.0). Phylogenetic analysis based on the complete aa sequence of RdRp further suggested that FeMV1 is a new member of the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus associated with F. equiseti.

RNA-seq Analysis of Rhizoctonia solani AG-4HGI Strain BJ-1H Infected by a New Viral Strain of Rhizoctonia solani Partitivirus 2 Reveals a Potential Mechanism for Hypovirulence.

Rhizoctonia solani partitivirus 2 (RsPV2), in the genus Alphapartitivirus, confers hypovirulence on R. solani AG-1-IA, the causal agent of rice sheath blight. In this study, a new strain of RsPV2 obtained from R. solani AG-4HGI strain BJ-1H, the causal agent of black scurf on potato, wasidentified and designated as Rhizoctonia solani partitivirus 2 strain BJ-1H (RsPV2-BJ). An RNA sequencing analysis of strain BJ-1H and the virus RsPV2-BJ-free strain BJ-1H-VF derived from strain BJ-1H was conducted to investigate the potential molecular mechanism of hypovirulence induced by RsPV2-BJ. In total, 14,319 unigenes were obtained, and 1,341 unigenes were identified as differentially expressed genes (DEGs), with 570 DEGs being down-regulated and 771 being up-regulated. Notably, several up-regulated DEGs were annotated to cell wall degrading enzymes, including ß-1,3-glucanases. Strain BJ-1H exhibited increased expression of ß-1,3-glucanase after RsPV2-BJ infection, suggesting that cell wall autolysis activity in R. solani AG-4HGI strain BJ-1H might be promoted by RsPV2-BJ, inducing hypovirulence in its host fungus R. solani AG-4HGI. To the best of our knowledge, this is the first report on the potential mechanism of hypovirulence induced by a mycovirus in R. solani.

Pre- and postharvest measures used to control decay and mycotoxigenic fungi in potato (Solanum tuberosum L.) during storage.

Potato (Solanum tuberosum L.), a worldwide, staple food crop, is susceptible to postharvest rots caused by a variety of fungal pathogens, including Fusarium spp., Alternaria spp., Phytophthora infestans, Helminthosporium solani, Rhizoctonia solani, and Colletotrichum coccodes. Rots resulting from infections by these pathogens cause a significant reduction in potato quality and marketable yield. Importantly, some of these decay fungi also produce mycotoxins that represent a potential risk to human health. In the present review, an overview and discussion are provided on the epidemiology and pathogenesis of decay fungi, especially Fusarium spp., that include recent data derived from genomic and phylogenetic analyses. The biosynthesis and functional role of fungitoxic metabolites such as trichothecene mycotoxins and fusaric acid, produced in rotted potatoes are also reviewed. Advances in pre- and postharvest measures for rot management, especially eco-friendly methods including physical control, biological control, the use of natural compounds, and other agricultural management practices are also reviewed. Lastly, novel approaches to control potato dry rot such as the use of mycoviruses and CRISPR technology are highlighted.

The Key Glutathione S-Transferase Family Genes Involved in the Detoxification of Rice Gramine in Brown Planthopper Nilaparvata lugens.

Phytochemical toxins are considered a defense measure for herbivore invasion. To adapt this defensive strategy, herbivores use glutathione S-transferases (GSTs) as an important detoxification enzyme to cope with toxic compounds, but the underlying molecular basis for GST genes in this process remains unclear. Here, we investigated the basis of how GST genes in brown planthopper (BPH, Nilaparvata lugens (Stål)) participated in the detoxification of gramine by RNA interference. For BPH, the LC25 and LC50 concentrations of gramine were 7.11 and 14.99 µg/mL at 72 h after feeding, respectively. The transcriptions of seven of eight GST genes in BPH were induced by a low concentration of gramine, and GST activity was activated. Although interferences of seven genes reduced BPH tolerance to gramine, only the expression of NlGST1-1, NlGSTD2, and NlGSTE1 was positively correlated with GST activities, and silencing of these three genes inhibited GST activities in BPH. Our findings reveal that two new key genes, NlGSTD2 and NlGSTE1, play an essential role in the detoxification of gramine such as NlGST1-1 does in BPH, which not only provides the molecular evidence for the coevolution theory, but also provides new insight into the development of an environmentally friendly strategy for herbivore population management.

Complete genome sequence of the first chrysovirus from the phytopathogenic fungus Alternaria solani on potato in China.

The complete genome sequence of a double-stranded RNA (dsRNA) mycovirus that was isolated from Alternaria solani strain DT-10 causing potato foliar disease was determined. The virus, designated as "Alternaria solani chrysovirus 1" (AsCV1), has four dsRNA segments (dsRNA 1-4) with a length of 3600 bp, 3128 bp, 2996 bp, and 2714 bp, respectively. The RNA-dependent RNA polymerase (RdRp, 1084 amino acids [aa]), putative capsid protein (905 aa), alphachryso-P3 (835 aa), and alphachryso-P4 (729 aa) were encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively, which had the highest sequence identity of 41.77%-72.38% to their counterparts in Helminthosporium victoriae virus 145S (HvV145S) of the genus Alphachrysovirus, family Chrysoviridae. Moreover, the 5'-untranslated regions (UTRs) of AsCV1 dsRNA 1-4, which contained several unique inserts (3-37 bp) and deletions (5-64 bp), shared 51.65%-68.01% identity with those of HvV145S. Phylogenetic analysis based on RdRp sequences showed that AsCV1 clustered the most closely with HvV145S. Considering its distinct host specificity, the low sequence similarity of its encoded proteins to those of other viruses, the unusual features of the 5'-UTRs of its dsRNA 1-4, and the phylogenetic position of its RdRp gene, AsCV1 should be considered a member of a new species in the genus Alphachrysovirus. To the best of our knowledge, this is the first alphachrysovirus identified from phytopathogenic A. solani.

Exogenous salicylic acid improves resistance of aphid-susceptible wheat to the grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae).

Salicylic acid (SA), a phytohormone, has been considered to be a key regulator mediating plant defence against pathogens. It is still vague how SA activates plant defence against herbivores such as chewing and sucking pests. Here, we used an aphid-susceptible wheat variety to investigate Sitobion avenae response to SA-induced wheat plants, and the effects of exogenous SA on some defence enzymes and phenolics in the plant immune system. In SA-treated wheat seedlings, intrinsic rate of natural increase (rm), fecundity and apterous rate of S. avenae were 0.25, 31.4 nymphs/female and 64.4%, respectively, and significantly lower than that in the controls (P < 0.05). Moreover, the increased activities of phenylalanine-ammonia-lyase, polyphenol oxidase (PPO) and peroxidase in the SA-induced seedlings obviously depended on the sampling time, whereas activities of catalase and 4-coumarate:CoA ligase were suppressed significantly at 24, 48 and 72 h in comparison with the control. Dynamic levels of p-coumaric acid at 96 h, caffeic acid at 24 and 72 h and chlorogenic acid at 24, 48 and 96 h in wheat plants were significantly upregulated by exogenous SA application. Nevertheless, only caffeic acid content was positively correlated with PPO activity in SA-treated wheat seedlings (P = 0.031). These findings indicate that exogenous SA significantly enhanced the defence of aphid-susceptible wheat variety against aphids by regulating the plant immune system, and may prove a potential application of SA in aphid control.

Chitosan induces resistance to tuber rot in stored potato caused by Alternaria tenuissima.

Alternaria tenuissima infects stored potatoes, and causes tuber rot, resulting in significant economic losses. As a naturally-occurring polysaccharide (poly-ß-(1 → 4) N-acetyl-D-glucosamine), chitosan has been reported to be an eco-friendly alternative to synthetic fungicides for the control of postharvest diseases on agricultural commodities. In this study, application of 0.25-1.25 g/L chitosan significantly inhibited spore germination and mycelial growth of A. tenuissima in vitro, with the greatest inhibitory effect observed at the highest concentration. Cytological and biochemical analysis of A. tenuissima spores indicated that exposure to 1.25 g/L chitosan significantly damaged the plasma membrane and increased the level of lipid oxidation. Gene expression analysis in potato tuber revealed that an application of 1.25 g/L chitosan induced the expression of defense-related genes, including catalase, peroxidase, polyphenol oxidase, chitinase and ß-1,3-glucanase, and the level of flavonoids and lignin. Chitosan effectively controlled tuber rot caused by A. tenuissima. Collectively, results of the current study indicate that the ability of chitosan to reduce Alternaria rot in stored potato tubers is due to its direct antifungal activity and its ability to induce defense responses in potato tuber tissues. Chitosan may have the potential as a substitute for synthetic fungicides to reduce postharvest losses in potato.

Interaction of Ferulic Acid with Glutathione S-Transferase and Carboxylesterase Genes in the Brown Planthopper, Nilaparvata lugens.

Plant phenolics are crucial defense phytochemicals against herbivores and glutathione S-transferase (GST) and carboxylesterase (CarE) in herbivorous insects are well-known detoxification enzymes for such xenobiotics. To understand relationship between a plant phenolic and herbivore GST or CarE genes, we evaluated the relationship between a rice phenolic ferulic acid and resistance to brown planthopper (BPH, Nilaparvata lugens), and investigated the interaction of ferulic acid with GST or CarE genes in BPH. The results indicate that ferulic acid content in tested rice varieties was highly associated with resistance to BPH. Bioassays using artificial diets show that the phenolic acid toxicity to BPH was dose dependent and the LC25 and LC50 were 5.81 and 23.30 µg/ml at 72 hr, respectively. Activities of the enzymes BPH GST and CarE were increased at concentrations below the LC50 of ferulic acid. Moreover, low ferulic acid concentrations (< LC25) upregulated the transcriptional levels of NlGSTD1 and NlGSTE1 of the GST family and NlCE of the CarE family. By using dsRNA-induced gene silencing (DIGS) of GST or CarE, it was shown that suppressed expression levels of NlGSTD1, NlGSTE1 and NlCE were 14.6%-21.2%, 27.8%-34.2%, and 10.5%-19.8%, respectively. Combination of NlGSTD1, NlGSTE1 or NlCE knockdown with ferulic acid increased nymph mortality by 92.9%, 119.9%, or 124.6%, respectively. These results suggest that depletion of detoxification genes in herbivorous insects by plant-mediated RNAi technology might be a new potential resource for improving rice resistance to BPH.

Exploring poisonous mechanism of honeybee, Apis mellifera ligustica Spinola, caused by pyrethroids.

As the important intracellular secondary messengers, calcium channel is the target of many neurotoxic pesticides as calcium homeostasis in the neuroplasm play important role in neuronal functions and behavior in insects. This study investigated the effect of deltamethrin (DM) on calcium channel in the brain nerve cells of adult workers of Apis mellifera ligustica Spinola that were cultured in vitro. The results showed that the intracellular calcium concentration was significantly elevated even with a very low concentration of the DM (3.125×10-2mg/L). Further testing revealed that T-type voltage-gated calcium channels (VGCCs), except for sodium channels, was one of the target of DM on toxicity of Apis mellifera, while DM has no significant effect on the L-type VGCCs, N-methyl-d-aspartate receptor-gated calcium channels and calcium store. These results suggesting that the DM may act on T-type VGCCs in brain cells of honeybees and result in behavioral abnormalities including swarming, feeding, learning, and acquisition.

Glutathione S-transferase of brown planthoppers (Nilaparvata lugens) is essential for their adaptation to gramine-containing host plants.

Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms.

Xenobiotic metabolism of plant secondary compounds in the English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae).

Plant secondary compounds have been documented to be deleterious to insects and other herbivores in diverse ways. In this study, the effect of catechol (phenolics), gramine (alkaloid) and L-ornithine-HCI (non-protein amino acid) on the activities of xenobiotic metabolizing enzymes in English grain aphid, Sitobion avenae, was evaluated. Phase I enzymes investigated in this study included carboxylesterase (CarE), and oxidoreductase, whereas Phase II enzymes were represented by glutathione S-transferase (GST). In general, CarE and GST activities in S. avenae were positively correlated with the concentration of plant secondary compounds in artificial diets. Oxidoreductase activity, however, displayed a different profile. Specifically, peroxidase (POD) and polyphenol oxidase (PPO) activities in S. avenae were positively correlated with concentrations of dietary catechol and gramine, respectively, whereas catalase (CAT) activity was significantly suppressed by the higher concentration of catechol, gramine and L-ornithine-HCl. These combined results suggest that CarE and GST in S. avenae are key enzymes to breakdown a broad spectrum of plant secondary compounds, whereas oxidoreductase, including PPO and POD, degrades specific groups of plant secondary compounds.

[Effects of plant hedgerow on population dynamics of wheat aphid and its natural enemies].

This paper studied the effects of planting different kinds of plant hedgerow (Amorpha fruticosa, Vetiveria zizanioides, Eulaliopsis binata, and Medicago sativa) on the population dynamics of wheat aphid and its natural enemies in the hillside wheat fields in Ziyang City of Sichuan Province, Southwest China. On the 20 degrees hillside, A. fruticosa hedgerow inhibited the occurrence of wheat aphid in the wheat field significantly, and the parasitoid densities were equal to or significantly lower than those in the wheat field with V. zizanioides hedgerow. On the 12 degrees hillside, M. sativa and E. binata hedgerows delayed the peak time of wheat aphid occurrence, and E. binata hedgerow suppressed the wheat aphid population density significantly. In the wheat field with M. sativa hedgerow, the parasitoid densities were significantly higher than those with no hedgerow. An equal or significantly higher ladybird density was observed in the field with M. sativa hedgerow. The olfactory responses showed that E. binata volatiles had repellent effect on both Sitobion avenae and Rhopalosiphum padi, but attracted a spider species of oxyopidae. M. sativa volatiles attracted S. avenae, whereas neither M. sativa nor E. binata volatiles caused obvious behavior response of ladybird adults. Therefore, planting A. fruticosa and E. binata as the hedgerows in hillside wheat fields could not only prevent the soil erosion from seasonal rainfall, but also benefit the control of pest insects.

Detoxification of gramine by the cereal aphid Sitobion avenae.

Secondary metabolites play an important role in host plant resistance to insects, and insects, in turn, may develop mechanisms to counter plant resistance mechanisms. In this study, we investigated the toxicity of gramine to the cereal aphid Sitobion avenae and some enzymatic responses of S. avenae to this alkaloid. When S. avenae fed on an artificial diet containing gramine, mortality occurred in a dose-dependent manner. The LC(50) of gramine was determined to be 1.248 mM. In response to gramine, S. avenae developed increased activities of carboxylesterase and glutathione S-transferase, two important detoxification enzymes. The activities of both enzymes were positively correlated with the concentration of dietary gramine. In addition, the activities of peroxidase and polypheolic oxidase, two important oxidoreductase enzymes in S. avenae, increased in response to gramine; however, catalase activity decreased when insects were exposed to higher levels of dietary gramine. The potential role of gramine in host plant resistance and S. avenae counter-resistance is discussed.

Constitutive and induced activities of defense-related enzymes in aphid-resistant and aphid-susceptible cultivars of wheat.

Phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) are considered important biochemical markers in host plant resistance against pest insects. Constitutive activity of these enzymes was analyzed in resistant and susceptible wheat cultivars against cereal aphid Sitobion avenae (F.) at various developmental stages, i.e., tillering, stem elongation, flag leaf, and ear. Following aphid infestation, the activity of these enzymes was determined at the flag leaf and ear stages. Resistant cultivars exhibited greater constitutive PAL activity than susceptible ones at the tillering, stem elongation, and flag leaf stages. Aphid infestation enhanced levels of PAL activity in the flag leaf and ear stages in both resistant and susceptible cultivars. Constitutive PPO activity was higher in the resistant cultivars at all developmental stages. Aphid infestation induced increases in PPO activity in the flag leaf and ear stages of one susceptible cultivar, whereas induction in resistant cultivars was weaker. Resistant cultivars showed greater constitutive POD activity in the tillering, stem elongation, and flag leaf stages, while aphid infestation induced POD activity in all cultivars, especially in susceptible ones. The potential role of PAL, PPO, and POD in wheat defense against aphid infestation is discussed.

[Dynamics of indole alkaloids in wheat varieties with various resistance to wheat aphid Sitobion avenae].

UV spectrometry measurement on the indole alkaloids (IA) contents of seven wheat varieties (KOK1679, My295, Li, Han4564 and Yanda1817, resistant to S. avenae; Shaanximazha and Lovrin10, susceptible to S. avenae) showed that during tillering and stem elongation stages, KOK1679, My295 and Li had a higher IA content (> 0.5 mg.g-1 FW); and during heading and anthesis stages, the penultimate leaves of all five resistant varieties contained higher IA (> 0.5 mg.g-1 FW). The IA content was lower in the flat leaves of both resistant and susceptible varieties, but was significantly higher in the ears of resistant varieties (up to 0.645 mg.g-1 FW in KOK1679) than in those of susceptible ones. It was showed that the IA content in wheat's ear played an important role in the resistance to S. avenae.