Leguminous green manure intercropping changes the soil microbial community and increases soil nutrients and key quality components of tea leaves.

Intercropping, a green and sustainable planting pattern, has demonstrated positive effects on plant growth and the soil environment. However, there is currently little research on the influence of intercropping leguminous plants and using them as green manure on the soil environment and tea quality. During the profuse flowering period of Chinese milkvetch, the contents of tea amino acids and soluble sugar in intercropping tea plants with soybean increased by 6.89 and 54.58%. Moreover, there was 27.42% increase in soil ammonium nitrogen and 21.63% increase in available nitrogen. When Chinese milkvetch was returned to soil for 1 month during its profuse flowering period, the soybean and Chinese milkvetch as green manure enhanced tea amino acids and soluble sugar by 9.11 and 33.96%, and soil ammonium nitrogen, nitrate nitrogen and available nitrogen increased by 25.04, 77.84, and 48.90%. Intercropping systems also have positive effects on tea quality components, soil fertility, and soil microbial communities during the profuse flowering period of soybeans and when soybeans with this period were returned to the field for 1 month. Furthermore, the soil fertility index was significantly increased, especially in the intercropping system of tea-soybean-Chinese milkvetch. The soil bacterial community complexity and fungal community interactions were significantly increased. Soil pH, nitrate nitrogen, and available phosphorus were found to be crucial influencing factors on soil microbial communities, specifically bacterial communities. These results highlight the significance of optimizing intercropping systems to improve the soil environment and tea quality components. They also provide a theoretical foundation for promoting the sustainable development of tea plantations.

Diamide-linked imidazolyl Poly(dicationic ionic liquid)s for the conversion of CO2 to cyclic carbonates under ambient pressure.

The ionic active centers and hydrogen-bond donors (HBDs) in heterogeneous catalytic materials are highly beneficial for enhancing the interaction between solid-liquid-gas three-phase interfaces and promoting effective fixation of carbon dioxide (CO2). Diamide-linked imidazolyl poly(dicationic ionic liquid)s catalysts PIMDILs (PMAIL-x and PBAIL-2) were synthesized through the copolymerization of diamide-linked imidazolyl dicationic ionic liquids (IMDILs) with divinylbenzene (DVB), which successfully enable the simultaneous construction of high-density and uniformly distributed ionic active centers (2.014-4.883 mmol g-1) and hydrogen-bond donors (HBDs). The as-synthesized PIMDILs present excellent catalytic activity in promoting the cycloaddition of CO2 with epoxides. PMAIL-2 could convert epichlorohydrin (ECH) with a quantitative conversion of 99.8 % (selectivity > 99 %) under ambient pressure. Furthermore, only a decrease in activity of 5 % was observed even after six cycles of recycling. The excellent conversions (>97.3 %) were achieved for various terminal substituted epoxides. The experimental and characterization results reveal that the high-density ionic active centers and amide HBDs can effectively activate the reaction substrates, their synergistic effect plays a crucial role at the catalyst interface. This work is expected to provide some useful insights for the rational construction of heterogeneous catalysts for CO2 conversion.

CsMYB Transcription Factors Participate in Jasmonic Acid Signal Transduction in Response to Cold Stress in Tea Plant (Camellia sinensis).

Low-temperature stress is an increasing problem for the cultivation of tea (Camellia sinensis), with adverse effects on plant growth and development and subsequent negative impacts on the tea industry. Methyl jasmonate (MeJA), as a plant inducer, can improve the cold-stress tolerance in tea plants. R2R3-MYB transcription factors (TFs) are considered potentially important regulators in the resistance to cold stress in plants. However, the molecular mechanisms, by which MYB TFs via the jasmonic acid pathway respond to cold stress in the tea plant, remain unknown. In this study, physiological and biochemical assays showed that exogenous MeJA application could effectively promote ROS scavenging in the tea plant under cold stress, maintaining the stability of the cell membrane. Sixteen R2R3-MYB TFs genes were identified from the tea plant genome database. Quantitative RT-PCR analysis showed that three CsMYB genes were strongly induced under a combination of MeJA and cold-stress treatment. Subcellular localization assays suggest CsMYB45, CsMYB46, and CsMYB105 localized in the nucleus. Exogenous MeJA treatment enhanced the overexpression of CsMYB45, CsMYB46, and CsMYB105 in E. coli and improved the growth and survival rates of recombinant cells compared to an empty vector under cold stress. Yeast two-hybrid and bimolecular fluorescence complementation experiments confirmed that CsMYB46 and CsMYB105 interacted with CsJAZ3, CsJAZ10, and CsJAZ11 in the nucleus. Taken together, these results highlight that CsMYB45, CsMYB46, and CsMYB105 are not only key components in the cold-stress signal response pathway but also may serve as points of confluence for cold stress and JA signaling pathways. Furthermore, our findings provide new insight into how MYB TFs influence cold tolerance via the jasmonic acid pathway in tea and provide candidate genes for future functional studies and breeding.

Comparative Metabolomics Analysis of Stigmas and Petals in Chinese Saffron (Crocus sativus) by Widely Targeted Metabolomics.

The dried stigmas of Crocus sativus, commonly known as saffron, are consumed largely worldwide because it is highly valuable in foods and has biological activities beneficial for health. Saffron has important economic and medicinal value, and thus, its planting area and global production are increasing. Petals, which are a by-product of the stigmas, have not been fully utilized at present. We compared the metabolites between the stigmas and petals of C. sativus using a non-targeted metabolomics method. In total, over 800 metabolites were detected and categorized into 35 classes, including alkaloids, flavonoids, amino acids and derivatives, phenols and phenol esters, phenylpropanoids, fatty acyls, steroids and steroid derivatives, vitamins, and other metabolites. The metabolite composition in the petals and stigmas was basically similar. The results of the study showed that the petals contained flavonoids, alkaloids, coumarins, and other medicinal components, as well as amino acids, carbohydrates, vitamins, and other nutritional components. A principal components analysis (PCA) and an orthogonal partial least-squares discriminant analysis (OPLS-DA) were performed to screen the different metabolic components. A total of 339 differential metabolites were identified, with 55 metabolites up-regulated and 284 down-regulated. The up-regulated metabolites, including rutin, delphinidin-3-O-glucoside, isoquercitrin, syringaresinol-di-O-glucoside, dihydrorobinetin, quercetin, and gallocatechin, were detected in the petals. The down-regulated metabolites were mainly glucofrangulin B, acetovanillone, daidzein, guaiazulene, hypaphorine, indolin-2-one, and pseudouridine. KEGG annotation and enrichment analyses of the differential metabolites revealed that flavonoid biosynthesis, amino acids biosynthesis, and arginine and proline metabolism were the main differentially regulated pathways. In conclusion, the petals of C. sativus are valuable for medicine and foods and have potential utility in multiple areas such as the natural spice, cosmetic, health drink, and natural health product industries.

Genome-Wide Analysis of the TCP Gene Family and Their Expression Pattern Analysis in Tea Plant (Camellia sinensis).

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors TEOSINTE BRANCHED1/CYCLOIDEA/PCF have been suggested to control the cell growth and proliferation in meristems and lateral organs. A total of 37 CsTCP genes were identified and divided into two classes, class I (PCF, group 1) and class II (CIN CYC/TB1, groups 2, and 3). The residues of TEOSINTE BRANCHED1/CYCLOIDEA/PCF of Camellia sinensis (Tea plant) (CsTCP) proteins between class I and class II were definitely different in the loop, helix I, and helix II regions; however, eighteen conserved tandem was found in bHLH. There are a large number of CsTCP homologous gene pairs in three groups. Additionally, most CsTCP proteins have obvious differences in motif composition. The results illuminated that CsTCP proteins in different groups are supposed to have complementary functions, whereas those in the same class seem to display function redundancies. There is no relationship between the number of CsTCP gene members and genome size, and the CsTCP gene family has only expanded since the divergence of monocots and eudicots. WGD/segmental duplication played a vital role in the expansion of the CsTCP gene family in tea plant, and the CsTCP gene family has expanded a lot. Most CsTCP genes of group 1 are more widely and non-specifically expressed, and the CsTCP genes of group 2 are mainly expressed in buds, flowers, and leaves. Most genes of group 1 and some genes of group 2 were up-/downregulated in varying degrees under different stress, CsTCP genes of group 3 basically do not respond to stress. TCP genes involved in abiotic stress response mostly belong to PCF group. Some CsTCP genes may have the same function as the homologous genes in Arabidopsis, but there is functional differentiation.

CsAAP7.2 is involved in the uptake of amino acids from soil and the long-distance transport of theanine in tea plants (Camellia sinensis L.).

Tea plant roots can uptake both inorganic nitrogen (NH4+ and NO3-) and organic nitrogen (amino acids) from the soil. These amino acids are subsequently assimilated into theanine and transported to young shoots through the xylem. Our previous study showed that CsLHT1 and CsLHT6 transporters take up amino acids from the soil, and CsAAPs participate in the transport of theanine. However, whether other amino acid transporters are involved in this process remains unknown. In this study, we identified two new CsAAPs homologous to CsAAP7, named CsAAP7.1 and CsAAP7.2. Heterologous expression of CsAAP7.1 and CsAAP7.2 in the yeast mutant 22Δ10α showed that CsAAP7.2 had the capacity to transport theanine and other amino acids, whereas CsAAP7.1 had no transport activity. Transient expression of the CsAAP7.2-GFP fusion protein in tobacco leaf epidermal cells confirmed its localization to the endoplasmic reticulum. Tissue-specific analysis showed that CsAAP7.2 was highly expressed in roots and stems. In addition, CsAAP7.2 overexpression lines were more sensitive to high concentrations of theanine due to the high accumulation of theanine in seedlings. Taken together, these findings suggested that CsAAP7.2 plays an important role in the uptake of amino acids from soil and the long-distance transport of theanine. These results provide valuable tools for nitrogen nutrition studies and enrich our understanding of theanine transport in tea plants.

CsCuAOs and CsAMADH1 Are Required for Putrescine-Derived γ-Aminobutyric Acid Accumulation in Tea.

Polyamines are a potential source of γ-aminobutyric acid (GABA) in plants under abiotic stress. However, studies on GABA enrichment in tea mostly focus on the GABA shunt, while the correlation between polyamine degradation and GABA formation in tea is largely unknown. In this study, tea plants responded to exogenous putrescine, resulting in a significant increase in GABA content, while the glutamate level did not change. At the same time, five copper-containing amine oxidase (CuAO) and eight aminoaldehyde dehydrogenase (AMADH) genes involved in the putrescine-derived GABA pathway were identified from the Tea Plant Information Archive. Expression analysis indicated that CsCuAO1, CsCuAO3 as well as CsAMADH1 were induced to play an important function in response to exogenous putrescine. Thus, the three genes were cloned and the catalytic efficiency of soluble recombinant proteins was determined. CsCuAOs and CsAMADH1 exhibited indispensable functions in the GABA production from putrescine in vitro. Subcellular localization assays indicated that CsAMADH1 was localized in plastid, while both CsCuAO1 and CsCuAO3 were localized in peroxisome. In addition, the synergistic effects of CsCuAOs and CsAMADH1 were investigated by a transient co-expression system in Nicotiana benthamiana. Our data suggest that these three genes regulate the accumulation of GABA in tea by participating in the polyamine degradation pathway and improve the content of GABA in tea to a certain extent. The results will greatly contribute to the production of GABA tea.

Distribution of trace metals in a soil-tea leaves-tea infusion system: characteristics, translocation and health risk assessment.

The effects of metal pollution on tea are of great concern to consumers. We apply Geographic information systems technology to study the distribution of heavy metal elements in tea plantation ecosystems in Jiangsu Province, explore the relationships among metals in the soil, tea leaves and tea infusions, and assess the human safety risks of metals. The concentrations of nine metals in a soil-tea leaves-tea infusion system were studied at 100 randomly selected tea plantations in Jiangsu Province, China. Concentrations of selected metals, zinc (Zn), nickel (Ni), manganese (Mn), chromium (Cr) and copper (Cu), were quantified using an inductively coupled plasma-optical emission spectrometer (ICP-OES), and cadmium (Cd), arsenic (As), plumbum (Pb) and mercury (Hg) were quantified using inductively coupled plasma-mass spectrometry (ICP-MS). Arc-Map 10.3 was used for the spatial analysis of metals in soil, tea leaves and tea infusions. We found that the contents of Mn, Ni and Zn are high level in soil, tea leaves and tea infusions. The Mn level showed a spatial distribution pattern with greater concentrations at the junction of Nanjing and Yangzhou, southwest of Changzhou and west of Suzhou. The hazard index (HI) values in north-central Nanjing, southern Suzhou, southwestern Changzhou and northern Lianyungang were relatively greater. The Zn, Ni, Mn, Cr and Cu levels in the soil-tea infusion system were 17.3, 45.5, 54.5, 1.5 and 14.3%, respectively. The order of the leaching rates of the elements was Ni > Cr > Zn > Mn > Cu. The relative contribution ratios of HI were in the order of Mn > Ni > Cu > Zn > Cr > Pb > Cd > As > Hg. In tea infusions, the Mn level has the greatest potential health risks to consumers. Moreover, using Csoil it was inferred that the safety thresholds of Zn, Ni, Mn, Cr and Cu in soil were 27,700, 50, 1230, 493,000 and 16,800 mg L-1, respectively. The content of heavy metals in soil and tea varies greatly in different regions of Jiangsu Province, 92% of the soil has heavy metal content that meets the requirements of pollution-free tea gardens, 91% of tea samples met the requirements of green food tea. The thresholds for Ni (50 mg L-1) and Mn (1230 mg L-1) can be used as maximum limits in tea plantation soils. The consumption of tea infusions did not pose metal-related risks to human health.

Tea plantation intercropping green manure enhances soil functional microbial abundance and multifunctionality resistance to drying-rewetting cycles.

Climate change leads to more serious drying-rewetting alternation disturbance, which furtherly affects soil ecosystem function and agriculture production. Intercropping green manure, as an ancient agricultural practice, can improve the physical, chemical, and biological fertility of soil in tea plantation. However, the effects of intercropping green manure on soil multifunctional resistance to drying-rewetting disturbance in tea plantation has not been reported. In this study, the effects of different green manure practices over four years (tea plant monoculture, tea plant and soybean intercropping, tea plant and soybean + milk vetch intercropping) on soil multifunctionality resistance to drying-rewetting cycles, and the pivotal influencing factors were investigated. We used quantitative PCR array and analysis of multiple enzyme activities to characterize the abundance of functional genes and ecosystem multifunctionality, respectively. Compared with tea plantation monoculture, tea plant intercropping soybean and soybean + milk vetch significantly increased multifunctionality resistance by 12.07% and 25.86%, respectively. Random forest analysis indicated that rather than the diversity, the abundance of functional genes was the major drive of multifunctionality resistance. The structure equation model further proved that tea plantation intercropping green manure could improve the abundance of C cycling related functional genes mediated by soil properties, and ultimately increased multifunctionality resistance to drying-rewetting disturbance. Therefore, tea plantation intercropping green manure is an effective approach to maintain the multifunctionality resistance, which is conducive to maintain the soil nutrient supply capacity and tea production under the disturbance of drying-rewetting alternation.

The effects of tea plants-soybean intercropping on the secondary metabolites of tea plants by metabolomics analysis.

BACKGROUND: Intercropping, especially with legumes, as a productive and sustainable system, can promote plants growth and improves the soil quality than the sole crop, is an essential cultivation pattern in modern agricultural systems. However, the metabolic changes of secondary metabolites and the growth in tea plants during the processing of intercropping with soybean have not been fully analyzed. RESULTS: The secondary metabolomic of the tea plants were significant influence with intercropping soybean during the different growth stages. Especially in the profuse flowering stage of intercropping soybean, the biosynthesis of amino acids was significantly impacted, and the flavonoid biosynthesis, the flavone and flavonol biosynthesis also were changed. And the expression of metabolites associated with amino acids metabolism, particularly glutamate, glutamine, lysine and arginine were up-regulated, while the expression of the sucrose and D-Glucose-6P were down-regulated. Furthermore, the chlorophyll photosynthetic parameters and the photosynthetic activity of tea plants were higher in the tea plants-soybean intercropping system. CONCLUSIONS: These results strengthen our understanding of the metabolic mechanisms in tea plant's secondary metabolites under the tea plants-soybean intercropping system and demonstrate that the intercropping system of leguminous crops is greatly potential to improve tea quality. These may provide the basis for reducing the application of nitrogen fertilizer and improve the ecosystem in tea plantations.

GABA shunt contribution to flavonoid biosynthesis and metabolism in tea plants (Camellia sinensis).

γ-Aminobutyric acid (GABA), a signal molecule, is regarded as the intersection node of carbon and nitrogen metabolism, and its contributions to flavonoid metabolism in tea plant growth and development remain unclear. The correlation between the GABA shunt and flavonoid metabolism in tea plants is worth to explore. Secondary metabolites and their correlations with the taste of tea soup made from tea plants (Camellia sinensis) during different seasons were investigated. Related secondary metabolites and transcript profiles of genes encoding enzymes in the GABA shunt, flavonoid pathway and polyamine biosynthesis were measured throughout the tea plant growth seasons and after exogenous GABA applications. In addition, the abundance of differentially expressed proteins was quantified after treatments with or without exogenous GABA. The tea leaves showed the highest metabolite concentrations in spring season. CsGAD, CsGABAT, CsSPMS, CsODC, CsF3H and CsCHS were found to be important genes in the GABA and anthocyanin biosynthesis pathways. GABA and anthocyanin concentrations showed a positive correlation, to some extent, CsF3H and CsCHS played important roles in the GABA and anthocyanin biosynthesis.

Validating the Strategic Deployment of Blackleg Resistance Gene Groups in Commercial Canola Fields on the Canadian Prairies.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus L.) production in western Canada. Crop scouting and extended crop rotation, along with the use of effective genetic resistance, have been key management practices available to mitigate the impact of the disease. In recent years, new pathogen races have reduced the effectiveness of some of the resistant cultivars deployed. Strategic deployment and rotation of major resistance (R) genes in cultivars have been used in France and Australia to help increase the longevity of blackleg resistance. Canada also introduced a grouping system in 2017 to identify blackleg R genes in canola cultivars. The main objective of this study was to examine and validate the concept of R gene deployment through monitoring the avirulence (Avr) profile of L. maculans population and disease levels in commercial canola fields within the Canadian prairies. Blackleg disease incidence and severity was collected from 146 cultivars from 53 sites across Manitoba, Saskatchewan, and Alberta in 2018 and 2019, and the results varied significantly between gene groups, which is likely influenced by the pathogen population. Isolates collected from spring and fall stubble residues were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm9, AvrLm10, AvrLm11, AvrLepR1, AvrLepR2, AvrLep3, and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR-based markers. The Simpson's evenness index was very low, due to two dominant L. maculans races (AvrLm2-4-5-6-7-10-11 and AvrLm2-5-6-7-10-11) representing 49% of the population, but diversity of the population was high from the 35 L. maculans races isolated in Manitoba. AvrLm6 and AvrLm11 were found in all 254 L. maculans isolates collected in Manitoba. Knowledge of the blackleg disease levels in relation to the R genes deployed, along with the L. maculans Avr profile, helps to measure the effectiveness of genetic resistance.

Genome-Wide Identification and Expression Analysis of the NRAMP Family Genes in Tea Plant (Camellia sinensis).

The natural resistant-associated macrophage protein (NRAMP) is a kind of integral membrane transporter which could function on a wide range of divalent metal ions in plants. Little is known about the NRAMP family in Camellia sinensis. In this study, 11 NRAMP genes were identified from the tea plant genome. Phylogenetic analysis showed that the 11 CsNRAMP proteins were split into two groups. The proteins of group 1 contained the conserved motif 6 (GQSSTxTG), while most proteins in group 2 (excepting CsNRAMP7 and CsNRAMP10) contained the conserved residues of motif 6 and motif 2 (GQFIMxGFLxLxxKKW). The number of amino acids in coding regions of 11 CsNRAMP genes ranged from 279-1373, and they contained 3-12 transmembrane domains. Quantitative RT-PCR analysis showed that G1 genes, CsNRAMP3, CsNRAMP4, and CsNRAMP5, were extraordinarily expressed in roots, while G2 genes showed higher expression levels in the stems and leaves. The expression levels of CsNRAMPs in roots and leaves were detected to assess their responses to Pb treatment. The results indicated that CsNRAMPs were differentially regulated, and they might play a role in Pb transportation of tea plant. Subcellular localization assay demonstrated that CsNRAMP2 and CsNRAMP5 fused proteins were localized in the plasma membrane. Overall, this systematic analysis of the CsNRAMP family could provide primary information for further studies on the functional roles of CsNRAMPs in divalent metal transportation in tea plants.

The Effect of Temperature on the Hypersensitive Response (HR) in the Brassica napus-Leptosphaeria maculans Pathosystem.

Temperature is considered one of the crucial environmental elements in plant pathological interactions, and previous studies have indicated that there is a relationship between temperature change and host-pathogen interactions. The objective of this research is to investigate the link between temperature and the incompatible interactions of the host and pathogen. In this study, two Leptosphaeria maculans isolates (HCRT75 8-1 and HCRT77 7-2) and two Brassica napus genotypes (Surpass400 and 01-23-2-1) were selected. The selected B. napus genotypes displayed intermediate and resistant phenotypes. The inoculated seedlings were tested under three temperature conditions: 16 °C/10 °C, 22 °C/16 °C and 28 °C/22 °C (day/night: 16 h/8 h). Lesion measurements demonstrated that the necrotic lesions from the 28 °C/22 °C treatment were enlarged compared with the other two temperature treatments (i.e., 16 °C/10 °C and 22 °C/16 °C). The results of expression analysis indicated that the three temperature treatments displayed distinct differences in two marker genes (PATHOGENESIS-RELATED (PR) 1 and 2) for plant defense and one temperature-sensitive gene BONZAI 1 (BON1). Additionally, seven dpi at 22 °C/16 °C appeared to be the optimal pre-condition for the induction of PR1 and 2. These findings suggest that B. napus responds to temperature changes when infected with L. maculans.

Genome-Wide Identification and Analysis of the Valine-Glutamine Motif-Containing Gene Family in Brassica napus and Functional Characterization of BnMKS1 in Response to Leptosphaeria maculans.

Proteins containing valine-glutamine (VQ) motifs play important roles in plant growth and development as well as in defense responses to both abiotic and biotic stresses. Blackleg disease, which is caused by Leptosphaeria maculans, is the most important disease in canola (Brassica napus) worldwide; however, the identification of Brassica napus VQs and their functions in response to blackleg disease have not yet been reported. In this study, we conducted a genome-wide identification and characterization of the VQ gene family in Brassica napus, including chromosome location, phylogenetic relations, gene structure, motif domain, synteny analysis, and cis-elements categorization of their promoter regions. To understand Brassica napus VQ gene function in response to blackleg disease, we overexpressed BnVQ7 (BnaA01g36880D, also known as the mitogen-activated protein kinase 4 substrate 1 [MKS1] gene) in a blackleg-susceptible canola variety, Westar. Overexpression of BnMKS1 in canola did not improve its resistance to blackleg disease at the seedling stage; however, transgenic canola plants overexpressing BnMKS1 displayed an enhanced resistance to L. maculans infection at the adult plant stage. Expression levels of downstream and defense marker genes in cotyledons increased significantly at the necrotrophic stage of L. maculans infection in the overexpression line of BnMKS1, suggesting that the salicylic acid- and jasmonic acid-mediated signaling pathways were both involved in the defense responses. Together, these results suggest that BnMKS1 might play an important role in defense against L. maculans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Leptosphaeria maculans Isolates Reveal Their Allele Frequency in Western Canada.

Blackleg, caused by Leptosphaeria maculans, is a major disease of canola in Canada, Australia, and Europe. For effective deployment of resistant varieties and disease management, it is crucial to understand the population structure of L. maculans. In this study, we analyzed L. maculans isolates from commercial fields in western Canada from 2014 to 2016 for the presence and frequency of avirulence (Avr) genes. A total of 1,584 isolates were examined for the presence of Avr genes AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, AvrLepR1, AvrLepR2, and AvrLmS via a set of differential host genotypes carrying known resistance genes and a PCR assay. Several Avr genes showed a higher frequency in the pathogen population, such as AvrLm6 and AvrLm7, which were present in >90% of isolates, whereas AvrLm3, AvrLm9, and AvrLepR2 showed frequencies of <10%. A total of 189 races (different combinations of Avr genes) were detected, with Avr-2-4-6-7-S, Avr-1-4-6-7, and Avr-2-4-6-7 as the three predominant races. When the effect of crop rotation was assessed, only a 3-year rotation showed a significantly higher frequency of AvrLm2 relative to shorter rotations. This study provides the information for producers to select effective canola varieties for blackleg management and for breeders to deploy new R genes in disease resistance breeding in western Canada.

Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.

Generation and Characterization of a Virulent Leptosphaeria maculans Isolate Carrying a Mutated AvrLm7 Gene Using the CRISPR/Cas9 System.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is the most important disease affecting canola (Brassica napus) crops worldwide. We employed the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to generate the mutant isolate umavr7 from a point mutation of the AvrLm7 coding region in a L. maculans isolate (UMAvr7). Reverse transcription PCR and transcriptome data confirmed that the AvrLm7 gene was knocked out in the mutant isolate. Pathogenicity tests indicated that umavr7 can cause large lesions on a set of Brassica differential genotypes that express different resistance (R) genes. Comparative pathogenicity tests between UMAvr7 (wild type) and umavr7 on the corresponding B. napus genotype 01-23-2-1 (with Rlm7) showed that umavr7 is a mutant isolate, producing large gray/green lesions on cotyledons. The pathogenicity of the mutant isolate was shifted from avirulent to virulent on the B. napus Rlm7 genotype. Therefore, this mutant is virulence on the identified resistant genes to blackleg disease in B. napus genotypes. Superoxide accumulated differently in cotyledons in response to infection with UMAvr7 and umavr7, especially in resistant B. napus genotype 01-23-2-1. Resistance/susceptibility was further evaluated on 123 B. napus genotypes with the mutant isolate, umavr7. Only 6 of the 123 genotypes showed resistance to umavr7. The identification of these six resistant B. napus genotypes will lead to further studies on the development of blackleg disease resistance through breeding and the identification of novel R genes.

Metabolomic Analyses Provide New Insights into Signaling Mechanisms for Nutrient Uptake by Lateral Roots of Pruned Tea Plant (Camellia sinensis).

Pruning is an important plant management practice in tea cultivation. However, the mechanism underlying the dynamics of nutrient uptake by roots of pruned tea is unknown. This study investigated the metabolic alterations in lateral roots of pruned tea to unveil the mechanism of nutrient uptake. Elemental analysis revealed that pruning significantly increases the uptake of nutrients by lateral roots. Metabolic profiling showed significant metabolic variations in lateral roots of pruned tea. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that flavonoid biosynthesis, phenylpropanoid biosynthesis, and amino acid metabolism were differentially regulated in lateral roots. Caffeine metabolism was significantly hindered, while ethylene signaling was significantly induced in lateral roots of pruned plants. In addition, intermediates in the tricarboxylic acid (TCA) cycle were upregulated, indicating high rates of the TCA cycle. Therefore, pathways related to phenylpropanoid biosynthesis, TCA cycle, ethylene biosynthesis, and metabolism of amino acids contribute to higher nutrient uptake by lateral roots of the tea plant.