Panax ginseng is one of the most important medicinal and edible plants with pharmacological compounds mainly concentrated in the roots. Although chemical transformations of ginseng active compounds have been studied, their biotransformation processes by beneficial microbes are less reported. This study aimed to reveal changes in the functional components in P. ginseng roots fermented by the golden flower fungus Aspergillus cristatus. P. ginseng roots were incubated with A. cristatus, and the final product was given the name "Golden Flower Chinese Ginseng (GFCG)." A high-performance liquid chromatography (HP-LC) and liquid chromatograph mass spectrometer (LC-MS) system revealed that fermentation by A. cristatus caused metabolite changes in GFCG and promoted the production of rare ginsenosides. Transcriptomic analysis demonstrated that more than 72% of significantly differentially expressed genes in A. cristatus showed a decrease during interaction with P. ginseng. In the meantime, transcription-related genes were suppressed, while translational and post-translational events were activated, suggesting a special role of the fungal microbe when it is colonizing medical and edible plants. Therefore, this study provides detailed chemical characterization of GFCG and the potential molecular mechanism underlying the biotransformation of P. ginseng.

Panax notoginseng, an important traditional Chinese medicine with a long history of clinical application, possesses significant pharmacological value. P. notoginseng quality improvement has been extensively studied by chemical transformation methods; however, the bio-transformation method with beneficial microbes remains relatively underexplored. In this study, we first fermented P. notoginseng roots with the golden flower fungus Aspergillus cristatus, and then high-performance liquid chromatography (HPLC) was performed to reveal ginsenoside alterations in fungal treatment. To elucidate the underlying mechanisms, integrated transcriptome and metabolome methods were used to identify the differentially expressed genes of A. cristatus and the differentially accumulated metabolites in P. notoginseng roots after fermentation. Our study demonstrated that the P. notoginseng pharmacological compounds were dramatically changed, and the content of several important characteristic saponins, including ginsenosides R1, Re, Rb1, Rd, and Rf, was significantly increased. Concurrently, several metabolites associated with medicinal function were also significantly increased. These data further revealed the molecular characteristics of the beneficial fungus A. cristatus upon fermentation on the medicinal plant P. notginseng by reprogramming and activating fungal transcripts in the ribosome and oxidative phosphorylation-related events. This study provides new insights into the molecular mechanisms underlying A. cristatus-mediated enhancement of bioactive constituents in P. notoginseng, and establish a biotechnological foundation for improving the quality of cultivated P. notoginseng roots by controlled fermentation.

Tea is one of the most popular healthy and non-alcoholic beverages worldwide. Tea anthracnose is a disease in tea mature leaves and ultimately affects yield and quality. Colletotrichum camelliae is a dominant fungal pathogen in the tea field that infects tea plants in China. The pathogenic factors of fungus and the susceptible factors in the tea plant are not known. In this work, we performed molecular and genetic studies to observe a cerato-platanin protein CcCp1 from C. camelliae, which played a key role in fungal pathogenicity. △CcCp1 mutants lost fungal virulence and reduced the ability to produce conidia. Transcriptome and metabolome were then performed and analysed in tea-susceptible and tea-resistant cultivars, Longjing 43 and Zhongcha 108, upon C. camelliae wild-type CCA and △CcCp1 infection, respectively. The differentially expressed genes and the differentially accumulated metabolites in tea plants were clearly overrepresented such as linolenic acid and linoleic acid metabolism, glycerophospholipid metabolism, phenylalanine biosynthesis and metabolism, biosynthesis of flavonoid, flavone and flavonol etc. In particular, the accumulation of jasmonic acid was significantly increased in the susceptible cultivar Longjing 43 upon CCA infection, in the fungal CcCp1 protein dependent manner, suggesting the compound involved in regulating fungal infection. In addition, other metabolites in the glycerophospholipid and phenylalanine pathway were observed in the resistant cultivar Zhongcha 108 upon fungal treatment, suggesting their potential role in defense response. Taken together, this work indicated C. camelliae CcCp1 affected the tea plant lipid metabolism pathway to promote disease while the lost function of CcCp1 mutants altered the fungal virulence and plant response.

Microbes can be used to improve the quality of fermented beverages and food. Colletotrichum camelliae is one of the most dominant fungi associated with tea leaves. In the present study, black tea produced from the autumn leaves of Yellowish Yinghong 9 was treated with C. camelliae urate oxidase (CcUOX) to determine whether this treatment could improve tea quality. The effects of recombinant CcUOX on several black tea quality parameters were assessed, such as liquor color, aroma, and levels of theabrownins, thearubigins, total polyphenols, catechins, caffeine, xanthine, and hypoxanthine. CcUOX treatment significantly affected the aroma composition and volatile compound profiles. Specifically, volatiles in the benzenoid/phenylpropanoid, fatty acid metabolism, and amino acid pathways were increased in CcUOX-treated black tea, whereas levels of volatiles in the terpineol and carotenoid pathways were reduced. This suggested that the balance of aroma constituents in Yellowish Yinghong 9 black tea was affected by treatment with CcUOX. The present study provides evidence that pre-treatment of black tea with a single enzyme from a common tea-associated microbe could significantly improve autumn tea quality, validating this method for use in further studies of tea improvement.

Panax ginseng Meyer is one of the most valuable plants and is widely used in China, while ginseng anthracnose is one of the most destructive diseases. Colletotrichum panacicola could infect ginseng leaves and stems and causes serious anthracnose disease, but its mechanism is still unknown. Here, transcriptome and metabolism analyses of the host leaves were conducted to investigate the ginseng defense response affected by C. panacicola. Upon C. panacicola infection, ginseng transcripts altered from 14 to 24 h, and the expression of many defense-related genes switched from induction to repression. Consequently, ginseng metabolites in the flavonoid pathway were changed. Particularly, C. panacicola repressed plant biosynthesis of the epicatechin and naringin while inducing plant biosynthesis of glycitin, vitexin/isovitexin, and luteolin-7-O-glucoside. This work indicates C. panacicola successful infection of P. ginseng by intervening in the transcripts of defense-related genes and manipulating the biosynthesis of secondary metabolites, which might have antifungal activities.

Background: Gray mold, caused by Botrytis cinerea, is one of the major fungal diseases in agriculture. Biological methods are preferred over chemical fungicides to control gray mold since they are less toxic to the environment and could induce the resistance to pathogens in plants. In this work, we try to understand if ginseng defense to B. cinerea could be induced by fungal hypovirulent strain ABcSpd1. BcSpd1 encodes Zn(II)2Cys6 transcription factor which regulates fungal pathogenicity and we recently reported ABcSpd1 mutants reduced fungal virulence. Methods: We performed transcriptomic analysis of the host to investigate the induced defense response of ginseng treated by B. cinerea ABcSpd1. The metabolites in ginseng flavonoids pathway were determined by UPLC-ESI-MS/MS and the antifungal activates were then performed. Results: We found that ABcSpd1 enhanced the ginseng defense response when applied to healthy ginseng leaves and further changed the metabolism of flavonoids. Compared with untreated plants, the application of ABcSpd1 on ginseng leaves significantly increased the accumulation of p-coumaric acid and myricetin, which could inhibit the fungal growth. Conclusion: B. cinerea ABcSpd1 could effectively induce the medicinal plant defense and is referred to as the biological control agent in ginseng disease management. (c) 2023 The Korean Society of Ginseng. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Tea is one of the most well-known, healthy beverages in the world. Tea plants produce caffeine as a secondary metabolite. Colletotrichum camelliae is one of the most important microbes frequently isolated from tea fields, and it causes anthracnose disease in tea plant. In the present work, we performed molecular microbiology and transcriptomic analyses of the C. camelliae - tea plant interaction to investigate the mechanism of fungal virulence and plant defense. Upon infection of tea plant with C. camelliae, we observed alterations in the expression of fungal transcripts, including those of many genes associated with caffeine metabolism, such as those encoding various transporters, xanthine dehydrogenase, and urate oxidase (UOX). In particular, the deletion of C. camelliae urate oxidase (CcUOX), which is involved in the caffeine metabolism pathway, reduced fungal tolerance to caffeine, and impaired fungal virulence. CcUOX is involved in caffeine metabolism by the degradation of uric acid contents. C. camelliaeΔCcUOX mutants impaired uric acid degradation in vivo. The CcUOX gene was cloned from C. camelliae, overexpressed in Escherichia coli, and the recombinant CcUOX protein displayed maximum activity at 30°C and a pH of 4.0. The recombinant CcUOX efficiently reduced uric acid in vitro suggesting a promising application in caffeine-contaminated environment management and in producing food with low purine contents to prevent uric acid related human diseases, such as hyperuricemia and gout.

Botrytis cinerea is a necrotrophic microbe that causes gray mold disease in a broad range of hosts. In the present study, we conducted molecular microbiology and transcriptomic analyses of the host–B. cinerea interaction to investigate the plant defense response and fungal pathogenicity. Upon B. cinerea infection, plant defense responses changed from activation to repression; thus, the expression of many defense genes decreased in Arabidopsis thaliana. B. cinerea Zn(II)2Cys6 transcription factor BcSpd1 was involved in the suppression of plant defense as ΔBcSpd1 altered wild-type B05.10 virulence by recovering part of the defense responses at the early infection stage. BcSpd1 affected genes involved in the fungal sclerotium development, infection cushion formation, biosynthesis of melanin, and change in environmental pH values, which were reported to influence fungal virulence. Specifically, BcSpd1 bound to the promoter of the gene encoding quercetin dioxygenase (BcQdo) and positively affected the gene expression, which was involved in catalyzing antifungal flavonoid degradation. This study indicates BcSpd1 plays a key role in the necrotrophic microbe B. cinerea virulence toward plants by regulating pathogenicity-related compounds and thereby suppressing early plant defense.

Background:Panax ginseng Meyer is one of the most valuable medicinal plants which is enriched in anti-microbe secondary metabolites and widely used in traditional medicine. Botrytis cinerea is a necrotrophic fungus that causes gray mold disease in a broad range of hosts. B. cinerea could overcome the ginseng defense and cause serious leaf and root diseases with unknown mechanism. Methods:We conducted simultaneous transcriptomic and metabolomic analysis of the host to investigate the defense response of ginseng affected by B. cinerea. The gene deletion and replacement were then performed to study the pathogenic gene in B. cinerea during ginseng - fungi interaction. Results:Upon B. cinerea infection, ginseng defense responses were switched from the activation to repression, thus the expression of many defense genes decreased and the biosynthesis of antifungal metabolites were reduced. Particularly, ginseng metabolites like kaempferol, quercetin and luteolin which could inhibit fungi growth were decreased after B. cinerea infection. B. cinerea quercetin dioxygenase (Qdo) involved in catalyzing flavonoids degradation and △BcQdo mutants showed increased substrates accumulation and reduced disease development. Conclusion:This work indicates the flavonoids play a role in ginseng defense and BcQdo involves in B. cinerea virulence towards the P. ginseng. B. cinerea promotes disease development in ginseng by suppressing of defense related genes expression and reduction of antifungal metabolites biosynthesis.

BACKGROUND:Tea, which is produced from new shoots of existing tea plants (Camellia sinensis), is one of the most popular, non-alcoholic, healthy beverages worldwide. Colletotrichum camelliae is one of the dominant fungal pathogens of tea. The interaction of C. camelliae with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. Currently, many studies characterizing resistance or virulence and aggressiveness use lesion size at the infection sites on the leaves to quantify the growth of the pathogen. However, this method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection.RESULTS:A quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed for the quantification of C. camelliae growth on tea plant. This method was based on the comparison of fungal DNA in relation to plant biomass. This assay was used to investigate the phenotypes of tea plant cultivars in response to C. camelliae infection. Two cultivars, Zhongcha 108 (ZC108) and Longjing 43 (LJ43), were tested with this method. ZC108 was previously reported as an anthracnose-resistant cultivar against C. camelliae, while LJ43 was susceptible. The traditional lesion measurement method showed that both cultivars were susceptible to a virulent strain of C. camelliae, while the qRT-PCR approach indicated that very little fungal growth occurred in the anthracnose-resistant cultivar ZC108. The observed results in this study were consistent with previously published research. In addition, the DNA-based real-time PCR method was applied for analysis of pathogenic differences in general C. camelliae isolates and among several Colletotrichum spp that infect tea.CONCLUSIONS:This study showed that the DNA-based qRT-PCR technique is rapid, highly sensitive and easily applicable for routine experiments and could be used in screening for resistant tea plant cultivars or to identify differences in pathogen aggressiveness within and among Colletotrichum species.

茶树是一种重要的经济作物,广泛种植于热带和亚热带地区.随着茶产品经济价值的提高,茶叶生产安全备受关注.炭疽菌是引起茶树病害的病原菌之一,严重影响茶叶产量和茶产品品质,威胁我国茶产业的良性发展.利用抗病品种是防治茶树病害最为经济、有效的措施.目前,茶树炭疽菌种名使用混乱,茶树与炭疽菌的互作研究也较为薄弱,而简单依靠药剂防治病害也对茶叶质量安全造成一定的影响.本文梳理并明确了危害茶树的炭疽菌种类;总结了现有的茶树和炭疽菌互作的研究成果,提出识别免疫(effector triggered immunity,ETI)模式是茶树防御炭疽菌的主要机制;对茶树病理学存在的问题进行了归纳并提出解决办法,并展望了深入开展茶树抗病机理研究的思路,以期为制定茶园绿色防控技术及选育茶树抗病良种提供科学参考.

The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.

启动子在调控基因转录中具有重要的作用,为探明茶树茉莉酸及挥发性绿叶气味合成途径关键基因脂氧合酶LOX的表达调控规律,应用染色体步移技术克隆了CsLOX3基因的5’侧翼序列1 050 bp.采用PLACE、PlantCARE在线启动子预测工具等分析表明:该序列包含启动子核心元件如TATA-box、CAAT-box,以及多种类型的激素应答作用元件、生物或非生物胁迫响应相关的元件,以及逆境胁迫相关的转录因子结合元件等.启动子上包含的这些顺式作用元件为深入研究茶树CsLOX3基因的表达调控提供了参考.

In a halotolerant fungus Aspergillus glaucus CCHA, several functional proteins with stress-tolerant activity have been studied, but no secretory enzymes have been identified yet. The unique GH5 cellulase candidate from A. glaucus, an endoglucanase termed as AgCMCase, was cloned, expressed in the Pichia pastoris system and the purified enzyme was characterized. A large amount of recombinant enzyme secreted by the P. pastoris GS115 strain was purified to homogeneity. The molecular weight of the purified endoglucanase is about 55.0 kDa. The AgCMCase exhibited optimum catalytic activity at pH 5.0 and 55 °C. However, it remained relatively stable at temperatures ranging from 45 to 80 °C and pH ranging from 4.0 to 9.0. In addition, it showed higher activity at extreme NaCl concentrations from 1.0 to 4.0 M, suggesting it is an enzyme highly stable under heat, acid, alkaline and saline conditions. To evaluate the catalytic activity of AgCMCase, the hydrolysis products of rice and corn straws were successfully studied. In conclusion, the AgCMCase is a thermostable and salt-tolerant cellulase with potential for industrial application.

Rapid and massive transcriptional reprogramming upon pathogen recognition is the decisive step in plant-phytopathogen interactions. Plant transcription factors (TFs) are key players in this process but they require a suite of other context-specific co-regulators to establish sensory transcription regulatory networks to bring about host immunity. Molecular, genetic and biochemical studies, particularly in the model plants Arabidopsis and rice, are continuously uncovering new components of the transcriptional machinery that can selectively impact host resistance toward a diverse range of pathogens. Moreover, detailed studies on key immune regulators, such as WRKY TFs and NPR1, are beginning to reveal the underlying mechanisms by which defense hormones influence the function of these factors. Here we provide a short update on such recent developments.

The large WRKY transcription factor family is mainly involved in regulating plant immune responses. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic processes towards Botrytis cinerea strain 2100 infection and is essential for resistance. In contrast to B. cinerea strain 2100, the strain B05.10 is virulent on wild-type (WT) Col-0 Arabidopsis plants highlighting the genetic diversity within this pathogen species. We analysed how early WRKY33-dependent responses are affected upon infection with strain B05.10 and found that most of these responses were strongly dampened during this interaction. Ectopic expression of WRKY33 resulted in complete resistance towards this strain indicating that virulence of B05.10, at least partly, depends on suppressing WRKY33 expression/protein accumulation. As a consequence, the expression levels of direct WRKY33 target genes, including those involved in the biosynthesis of camalexin, were also reduced upon infection. Concomitantly, elevated levels of the phytohormone abscisic acid (ABA) were observed. Molecular and genetic studies revealed that ABA negatively influences defence to B05.10 and effects jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) levels. Susceptibility/resistance was determined by the antagonistic effect of ABA on JA, and this crosstalk required suppressing WRKY33 functions at early infection stages. This indicates that B. cinerea B05.10 promotes disease by suppressing WRKY33-mediated host defences.

[Objectives] The infection rates and taxonomic relationships between Wolbachia strains in the sister species Ectropis obliqua and E. grisescens (Lepidoptera: Geometridae) were identified by molecular methods in order to reveal the potential effects of this endosymbiont on these species. [Methods] Wolbachia infection in three populations of E. obliqua (Hangzhou and Yuhang, Zhejiang province; Wuxi, Jiangsu Province) and three populations of E. grisescens (Xinchang, Zhejiang Province; Xishui, Hubei Province; and Nanchang, Jiangxi Province) were detected using PCR with three specific primers for Wolbachia 16S rRNA and the Wolbachia ftsZ and wsp genes. All Wolbachia 16S rRNA, ftsZ and wsp genes from the three E. grisescens populations and some Wolbachia 16S rRNA and wsp genes from the three E. obliqua populations, were sequenced and analyzed. [Results] All E. obliqua and E. grisescens populations were infected with Wolbachia. Infection rates of the three E. grisescens populations were all 100%, but ranged from 22% to 95% among the three E. obliqua populations, with positive PCRs showing only weak bands in the electrophoretogram. There were no intra- and interspecific differences in the wsp sequences of E. obliqua and E. grisescens, however, intra- and interspecific differences in 16S rRNA sequences were between 0.362% and 0.727%, respectively. There was a 1.647% difference in two ftsZ haplotypes from E. grisescens, moreover, we were unable to successfully amplify ftsZ from all E. obliqua individuals. A phylogenetic tree based on the 16S rRNA and wsp sequences showed that all strains of Wolbachia detected belong to the Pip subgroup of the B group. [Conclusion] All three E. obliqua and E. grisescens populations tested are infected with Wolbachia strains belonging to the Pip subgroup of the B group, but the infection rate varies significantly between E. obliqua and E. grisescens. These results provide the basis for future study of the effects of Wolbachia on the biology and ecology of E. obliqua and E. grisescens.

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity.