Comparative physiological and transcriptome analysis provide insights into the inhibitory effect of osthole on Penicillium choerospondiatis.

Blue mold induced by Penicillium choerospondiatis is a primary cause of growth and postharvest losses in the fruit of Phyllanthus emblica. There is an urgent need to explore novel and safe fungicides to control this disease. Here, we demonstrated osthole, a natural coumarin compound isolated from Cnidium monnieri, exhibited a strong inhibitory effect on mycelia growth, conidial germination rate and germ tube length of P. choerospondiatis, and effectively suppressed the blue mold development in postharvest fruit of P. emblica. The median effective concentration of osthole was 9.86 mg/L. Osthole treatment resulted in cellular structural disruption, reactive oxygen species (ROS) accumulation, and induced autophagic vacuoles containing cytoplasmic components in fungal cells. Transcriptome analysis revealed that osthole treatment led to the differentially expressed genes mainly enriched in the cell wall synthesis, TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation. Moreover, osthole treatment led to increase genes expression involved in peroxisome, autophagy and endocytosis. Particularly, the autophagy pathway related genes (PcATG1, PcATG3, PcATG15, PcATG27, PcYPT7 and PcSEC18) were prominently up-regulated by osthole. Summarily, these results revealed the potential antifungal mechanism of osthole against P. choerospondiatis. Osthole has potentials to develop as a natural antifungal agent for controlling blue mold disease in postharvest fruits.

First report of Botryosphaeria dothidea causing fruit rot on Chinese olive (Canarium album Raeusch.) in Guangdong province of China.

Chinese olive (Canarium album Raeusch.) is a traditional Chinese medicinal plant, mainly cultivated in Guangdong and Fujian provinces in China (Lai et al. 2022). In October 2023, Chinese olive fruit spots were observed in all the Chinese olive orchards surveyed in Chaozhou city (23.75°N, 116.67°E) of Guangdong, with an incidence up to 15%. Early disease symptoms on fruits appeared as circular or irregular, dark brown to black spots with yellowish lesions, and later the spots slowly coalesced to form large necrotic areas, which seriously affected the fruit marketability. To isolate the causal agent, small pieces (~0.3 mm2) of fruit tissue were excised from the lesion margins, and surface-disinfested with 75% (v/v) ethanol for 1 min, followed by 1% NaClO for 3 min, and rinsed three times with sterile water. The pieces were then placed on potato-dextrose-agar (PDA) and incubated at 27°C. Ultimately, four fungal isolates were obtained with similar morphology phenotypes, colonies initially appeared white with irregular margins and after 4-6 days turned dark gray gradually with dense aerial myceliu. Microscopy revealed conidia were single-celled, hyaline, aseptate, fusiform to subclavate, and measured 18.1-22.5 µm × 6.4-9.3 µm (19.8 × 7.4 m on average, n = 30), which were consistent with those descriptions of Botryosphaeria dothidea (Vasic et al. 2013; Zhang et al. 2023). To further identity the isolates, partial sequences of ribosomal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin (TUB2) genes were amplified using primers ITS1/ITS5, TEF-F/R, TUB2-F/R, respectively (Xu et al., 2023; Hong et al. 2006). The sequences of four isolates were identical, and those of representative strain GDCZ-1 were deposited in GenBank (ITS, OR584295; TEF1-α, OR685157; TUB2, OR685158). Using Neighbor-Joining algorithm, phylogenetic tree based on concatenated sequences of ITS, TEF1-α, and TUB2 showed that GDCZ-1 clustered with B. dothidea. To fulfill Koch's postulates, pathogenicity tests were performed on healthy Chinese olive fruits using the needle-prick inoculation method. The fruits were wounded with a sterile needle at the equatorial area (depth of 3-4 mm), and inoculated with 10 µL of spore suspension (106 /mL). The control fruits were inoculated with sterile water. Inoculated fruits were placed in sterile plastic containers to maintain high relative humidity (almost 100%) and incubated at 27°C. After 4 days, the inoculated fruits showed similar symptoms with those observed in the field infected fruits, while the control remained asymptomatic. Pathogen re-isolated from the inoculated fruits showed identical morphological characteristics to the original isolate GDCZ-1. As far as we know, fruit rot caused by Alternaria alternata has been recently reported on C. album in China (Shao et al. 2024). To our knowledge, this is the first report of B. dothidea causing fruit rot disease on C. album in Guangdong. Our report will provide crucial information for studying the epidemiology and management of this disease.

The botanical eurycomanone is a potent growth regulator of the diamondback moth.

Eurycomanone is a quassinoid compound that is derived from Eurycoma longifolia, and it is often used as an indicator to evaluate the active ingredients of Eurycoma longifolia. However, Eurycomanone has rarely been reported to have biological activity toward pests. In this study, we evaluated the antifeedant activity of eurycomanone against the diamondback moth(Plutella xylostella), with a non-selective AFC50(the concentration that corresponds to 50% antifeedant action) value and selective AFC50 of 17.5 mg/L and 14.2 mg/L, respectively, which were 2.1-fold (36.9 mg/L) and 2-fold (28.5 mg/L) lower than that of azadirachtin, respectively. In addition, eurycomanone was used to treat the roots of Brassica chinensis L. at a concentration of 100 µg/g for 72 h. The antifeedant index was found to reach 93% by tracking the leaves. After feeding with 20 µg/g eurycomanone, no pupae or eclosion were observed. To explore this mechanism, we used scanning electron microscopy to discover that eurycomanone could prevent the development of taste receptors on the maxillary palp of diamondback moth larvae. Additional electrophysiological measurements showed that eurycomanone exhibited excitatory action to the central taste neurons of diamondback moth and significantly inhibited the GABAA receptor current. Eurycomanone exhibited significant activity as an antifeedant, inhibited growth and excelled at systemic absorption.

The stereoisomeric Bacillus subtilis HN09 metabolite 3,4-dihydroxy-3-methyl-2-pentanone induces disease resistance in Arabidopsis via different signalling pathways.

BACKGROUND: Plant immune responses can be induced by plant growth-promoting rhizobacteria (PGPRs), but the exact compounds that induce resistance are poorly understood. Here, we identified the novel natural elicitor 3,4-dihydroxy-3-methyl-2-pentanone from the PGPR Bacillus subtilis HN09, which dominates HN09-induced systemic resistance (ISR). RESULTS: The HN09 strain, as a rhizobacterium that promotes plant growth, can induce systemic resistance of Arabidopsis thaliana plants against Pseudomonas syringae pv. tomato DC3000, and the underlying role of its metabolite 3,4-dihydroxy-3-methyl-2-pentanone in this induced resistance mechanism was explored in this study. The stereoisomers of 3,4-dihydroxy-3-methyl-2-pentanone exhibited differential bioactivity of resistance induction in A. thaliana. B16, a 1:1 mixture of the threo-isomers (3R,4S) and (3S,4R), was significantly superior to B17, a similar mixture of the erythro-isomers (3R,4R) and (3S,4S). Moreover, B16 induced more expeditious and stronger callose deposition than B17 when challenged with the pathogen DC3000. RT-qPCR and RNA-seq results showed that B16 and B17 induced systemic resistance via JA/ET and SA signalling pathways. B16 and B17 activated different but overlapping signalling pathways, and these compounds have the same chemical structure but subtle differences in stereo configuration. CONCLUSIONS: Our results indicate that 3,4-dihydroxy-3-methyl-2-pentanone is an excellent immune elicitor in plants. This compound is of great importance to the systemic resistance induced by HN09. Its threo-isomers (3R,4S) and (3S,4R) are much better than erythro-isomers (3R,4R) and (3S,4S). This process involves SA and JA/ET signalling pathways.

Azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae by releasing cathepsin in the midgut.

Azadirachtin, a botanical insecticide with high potential, has been widely used in pest control. Azadirachtin has shown strong biological activity against Bactrocera dorsalis in toxicological reports, but its mechanism remains unclear. This study finds that azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae. The larval weights and body sizes of the azadirachtin-treated group were significantly less than those of the control group in a concentration-dependent manner. Further, pathological sections revealed that azadirachtin destroyed the midgut cell structure and intestinal walls, while TUNEL staining showed that azadirachtin could induce apoptosis of midgut cells, and Western blot analysis indicated that Bcl-XL expression was inhibited and cytochrome c (CytC) released into the cytoplasm. The results also imply azadirachtin-induced structural alterations in the Bactrocera dorsalis larvae midgut by activation of apoptosis. RNA-seq analysis of midgut cells found that 482 and 708 unique genes were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were enriched in apoptotic and lysosomal signaling pathways and included 26 genes of the cathepsin family. qRT-PCR verified the expression patterns of some DEGs, indicating that Cathepsin F was upregulated by 278.47-fold and that Cathepsin L and Cathepsin D were upregulated by 28.06- and 8.97-fold, respectively. Finally, association analysis between DEGs and DEMs (differentially expressed metabolites) revealed that azadirachtin significantly reduced the digestion and absorption of carbohydrates, proteins, fats, vitamins and minerals in the midgut. In conclusion, azadirachtin induces the release of cathepsin from lysosomes, causing apoptosis in the midgut. Ultimately, this leads to reduced digestion and absorption of nutrient metabolites in the midgut and inhibition of the growth and development of Bactrocera dorsalis larvae.

Comparative Analyses of the Transcriptome and Proteome of Comte de Paris and Smooth Cayenne to Improve the Understanding of Ethephon-Induced Floral Transition in Pineapple.

BACKGROUND/AIMS: Ethylene is usually used to induce floral transition in pineapple. However, its successful induction in plants categorized as Cayenne is difficult or completely ineffective, and information concerned is limited. The present study was undertaken to investigate the molecular mechanisms underlying this obstacle. METHODS: Transcriptome and proteome comparative analyses were performed to explore the important regulation and pathway variations after ethephon induction in the induction-easy 'Comte de Paris' (CP) and induction-hard 'Smooth Cayenne' (SC) cultivars via RNA-seq (RNA-sequencing) and iTRAQ (isobaric tags for relative and absolute quantification). RESULTS: CP and SC exhibited basic differences at the transcriptomic and proteomic levels before ethephon treatment, including the expression of genes and proteins related to ethylene signal transduction. After ethephon induction, the expression of genes and proteins involved in plant ethylene signal transduction and carbohydrate metabolism responded more strongly in CP than in SC. The expression of the floral meristem identity (FMI) genes AG, TFL and FT exhibited greater changes in CP, and more transcription factors responded in SC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that many differentially expressed genes (DEGs) in CP were annotated to terms and pathways involved in photoperiodism and shared components involved in carbohydrate metabolism and plant hormone signal transduction. CONCLUSION: These findings contribute to the understanding of the molecular mechanism underlying the variation between CP and SC in response to ethephon-mediated floral induction.

Growth inhibition and differences in protein profiles in azadirachtin-treated Drosophila melanogaster larvae.

Azadirachtin A is a very effective biopesticide widely used in insect pest control. It has strong antifeeding and growth inhibitory activity against most insects, however, its mode of action is still unclear. Proteomic experiments using 2DE indicate significant effects of Azadirachtin A on the amount of proteins related to growth inhibition in Drosophila melanogaster larvae. Twenty-one spots with different intensity in azadirachtin-treated larvae were identified. These proteins are involved in cytoskeletal organization, transcription and translation, hormonal regulation, and energy metabolism. Protein network analysis reveals heat shock protein 23 to be a potential target of azadirachtin. These results provide new insights into understanding the mechanism of growth inhibition in insects in response to azadirachtin.

Near-freezing temperature suppresses avocado (Persea americana Mill.) fruit softening and chilling injury by maintaining cell wall and reactive oxygen species metabolism during storage.

To enhance the postharvest quality of avocado (Persea americana Mill.) fruit, this study investigates alterations in cell wall metabolism and reactive oxygen species (ROS) metabolism during near-freezing temperature (NFT) storage, and explores their impact on fruit softening. The fruit was stored at 25 °C, 5 °C, 2 °C, and NFT, respectively. NFT storage retarded firmness loss and chilling injury in comparison with 25 °C, 5 °C, and 2 °C. NFT storage delayed the decrease of ionic-soluble pectin (ISP) and cellulose (CLL) contents by suppressing cell wall degradation enzyme activities. Correlation analysis showed that cell wall degradation enzyme activities were positively correlated to rates of ethylene release and respiration. Moreover, NFT storage maintained higher levels of DPPH and ABTS scavenging abilities, activities of superoxide dismutase, peroxidase, and catalase, as well as ascorbate-glutathione cycle (ascorbic acid, glutathione, glutathione disulfide, ascorbate peroxidase, cycle-related enzymes), thereby inhibited the increase of ROS content, malondialdehyde content, and cell membrane permeability. Fruit firmness and chilling injury were correlated with the contents of hydrogen (H2O2), superoxide anion (O2.-), ISP, and CLL. These results suggested that NFT could suppress fruit softening and chilling injury by inhibiting cell wall degradation through delaying respiration and ethylene production and suppressing ROS production via activation of antioxidant systems, thereby maintaining quality and prolonged storage life during avocado fruit storage.

Eurycomanone (EN) Activates Transcription Factor FoxO by Inhibiting the Insulin Signaling Pathway to Suppress the Development of Spodoptera frugiperda.

The insulin-like signaling (IIS) pathway is essential for insect growth and development. In this study, we showed that eurycomanone (EN) is an active compound with growth inhibitory activity against Spodoptera frugiperda larvae. Experiments in cells and RNA-seq analysis in the midgut showed that EN targeted the IIS pathway in S. frugiperda to activate the transcription factor SfFoxO (S. frugiperda forkhead boxO) to regulate mRNA levels associated with nutrient catabolism. Additionally, mass spectrometry imaging revealed that EN was distributed in the larval gut and enriched in the inner membrane of the gut. Immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EN induced program cell death (PCD) in the larvae midgut. Thus, EN targeted the insulin receptor to inhibit the IIS signaling pathway, exerting inhibitory activity on the growth and development of S. frugiperda larvae. Our results suggest that EN has great potential as a botanical pesticide, and the IIS signaling pathway may be an effective target for botanical pesticides.

Induction of Autophagy and Apoptosis via PI3K/AKT/TOR Pathways by Azadirachtin A in Spodoptera litura Cells.

Azadirachtin is one of the most effective botanical insecticides and has been widely used in pest control. Toxicological reports show that azadirachtin can induce apoptosis in various insect cell lines. However, studies of azadirachtin-induced autophagy in cultured insect cells are lacking. This study reports that azadirachtin A significantly inhibits cell proliferation by inducing autophagic and apoptotic cell death in Spodoptera litura cultured cell line (SL-1 cell). Characteristic autophagolysosome and Atg8-PE (phosphatidylethanolamine) accumulation were observed by electron microscopy and western blotting, indicating that azadirachtin triggered autophagy in SL-1 cell. Furthermore, azadirachtin inhibited survival signaling by blocking the activation of PI3K, AKT and the down-stream target of rapamycin. Similar to the positive control of starvation, azadirachtin induced the activation of insulin receptor (InR) via a cellular feedback mechanism. In addition, the autophagy-related 5 (Atg5), a molecular switch of autophagy and apoptosis, was truncated (tAtg5) to trigger cytochrome c release into the cytoplasm under azadirachtin stress, which indicated that azadirachtin induced apoptosis through autophagy. Our findings suggest that azadirachtin primarily induced autophagy in SL-1 cell by dysregulating InR- and PI3K/AKT/TOR pathways, then stimulated apoptosis by activating tAtg5.

Gene expression profile change and growth inhibition in Drosophila larvae treated with azadirachtin.

Azadirachtin is a botanical insecticide that affects various biological processes. The effects of azadirachtin on the digital gene expression profile and growth inhibition in Drosophila larvae have not been investigated. In this study, we applied high-throughput sequencing technology to detect the differentially expressed genes of Drosophila larvae regulated by azadirachtin. A total of 15,322 genes were detected, and 28 genes were found to be significantly regulated by azadirachtin. Biological process and pathway analysis showed that azadirachtin affected starch and sucrose metabolism, defense response, signal transduction, instar larval or pupal development, and chemosensory behavior processes. The genes regulated by azadirachtin were mainly enriched in starch and sucrose metabolism. This study provided a general digital gene expression profile of dysregulated genes in response to azadirachtin and showed that azadirachtin provoked potent growth inhibitory effects in Drosophila larvae by regulating the genes of cuticular protein, amylase, and odorant-binding protein. Finally, we propose a potential mechanism underlying the dysregulation of the insulin/insulin-like growth factor signaling pathway by azadirachtin.

Azadirachtin blocks the calcium channel and modulates the cholinergic miniature synaptic current in the central nervous system of Drosophila.

BACKGROUND: Azadirachtin is a botanical pesticide, which possesses conspicuous biological actions such as insecticidal, anthelmintic, antifeedancy, antimalarial effects as well as insect growth regulation. Deterrent for chemoreceptor functions appears to be the main mechanism involved in the potent biological actions of Azadirachtin, although the cytotoxicity and subtle changes to skeletal muscle physiology may also contribute to its insecticide responses. In order to discover the effects of Azadirachtin on the central nervous system (CNS), patch-clamp recording was applied to Drosophila melanogaster, which has been widely used in neurological research. RESULTS: Here, we describe the electrophysiological properties of a local neuron located in the suboesophageal ganglion region of D. melanogaster using the whole brain. The patch-clamp recordings suggested that Azadirachtin modulates the properties of cholinergic miniature excitatory postsynaptic current (mEPSC) and calcium currents, which play important roles in neural activity of the CNS. The frequency of mEPSC and the peak amplitude of the calcium currents significantly decreased after application of Azadirachtin. CONCLUSION: Our study indicates that Azadirachtin can interfere with the insect's CNS via inhibition of excitatory cholinergic transmission and partly blocking the calcium channel.

A proteomic approach provides new insights into the control of soil-borne plant pathogens by Bacillus species.

Beneficial microorganisms (also known as biopesticides) are considered to be one of the most promising methods for more rational and safe crop management practices. We used Bacillus strains EU07, QST713 and FZB24, and investigated their inhibitory effect on Fusarium. Bacterial cell cultures, cell-free supernatants and volatiles displayed varying degrees of suppressive effect. Proteomic analysis of secreted proteins from EU07 and FZB24 revealed the presence of lytic enzymes, cellulases, proteases, 1,4-ß-glucanase and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum. Our findings provide new knowledge on the mechanism of action of Bacillus species and insight into biocontrol mechanisms.