LbCOPT1 is a copper transporter induced in Lycium barbarum mycorrhizal roots, which allows tobacco with improved growth and nutrient uptake.

KEY MESSAGE: Overexpressing the copper transporter LbCOPT1 leads to a notable increase in the abundance of mycorrhizal arbuscules that suggests the potential application of LbCOPT1 in breeding programs aimed at enhancing symbiotic nutrient uptake in Lycium barbarum L.

Endophytic fungi of Lycium barbarum: isolation, determination, bioactivity and separation of compounds.

Lycium barbarum is widely distributed in China and used as a traditional Chinese medicine herb to treat dizziness, abdominal pain, dry cough, headache and fatigue. Several studies have examined the endophytes of L. barbarum from northwest China; however, few have focused on that from eastern China. The objective of this study was to isolate and determine the endophytic fungi of L. barbarum from Shandong province, as well as to obtain and identify active secondary metabolites from the endophytes. In this study, 17 endophytic fungi were isolated from L. barbarum and denoted as GQ-1 to GQ-17, respectively. These fungi were further classified into ten genera based on the morphological and ITS identification. The crude extracts of these fungi were obtained by using liquid fermentation and EtOAc extraction, and their antibacterial, cytotoxic, and antioxidant activities were evaluated. The results showed that GQ-6 and GQ-16 exhibited high inhibitory activity; GQ-6 and GQ-9 showed high cytotoxic activity and GQ-5 exhibited high scavenging capability for DPPH free radicals. Additionally, Cladosporium sp. GQ-6 was used to investigate the secondary metabolites. The crude extracts were purified by using column chromatography, reverse column, and liquid chromatography, and four monomeric compounds were identified, including two known compounds (α-acetylorcinol (1) and cladosporester B (2)) and two new compounds (cladosporacid F (3) and cladosporester D (4)). The anti-fungal and antibacterial activities of these compounds were confirmed, but no cytotoxic activity was observed. In conclusion, endophytic fungi of L. barbarum from eastern China can serve as a potential source of active natural products with antibacterial and antioxidant properties.

Fusaricide is a Novel Iron Chelator that Induces Apoptosis through Activating Caspase-3.

Nonsmall cell lung cancer (NSCLC) has been a fatal and refractory disease worldwide. Novel therapeutic developments based on fundamental investigations of anticancer mechanisms underlie substantial foundations to win the fight against cancer diseases. In this study, we isolated a natural product fusaricide (FCD) from an endophytic fungus of Lycium barbarum, identified as Epicoccum sp. For the first time, we discovered that FCD potently inhibited proliferation in a variety of human NSCLC cell lines, with relatively less toxicity to normal cells. Our study exhibited that FCD induced apoptosis, caused DNA damage and cell cycle arrest in G0/G1 phase, and activated caspase-3 as well as other apoptosis-related factors in human NSCLC NCI-H460 cells. FCD was proven to be an iron chelator that actively decreased levels of cellular labile iron pool in NCI-H460 cells in our study. FeCl3 supplement reversed FCD-induced apoptosis. The upregulation of transferrin receptor 1 (TfR1) and downregulation of ferritin heavy chain (FTH) expression were observed after FCD treatment. In summary, our study highlighted the potential anticancer effects of FCD against human NSCLCs and demonstrated that the FCD-mediated apoptosis depended on binding to intracellular iron.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Streptomyces lycii sp. nov., an endogenous actinomycete isolated from Lycium ruthenicum.

A novel endogenous actinobacteria strain, designated TRM 66187T, was isolated from Lycium ruthenicum sampled at Alar, Xinjiang, Northwest PR China, and characterized using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain TRM 66187T with the genus Streptomyces. The whole-cell sugar pattern of TRM 66187T consisted of galactose, glucose and ribose. The predominant menaquinones were MK-9(H4) and MK-9(H6). Major cellular fatty acids were iso-C14:0, iso-C15:0, anteiso-C15:0 and anteiso-C16:0. The detected polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and two unidentified polar lipids. The G+C content of strain TRM 66187T was 71.8 mol%. Results of phylogenetic analysis showed that strain TRM 66187T had 98.48% 16S rRNA gene sequence similarity to the closest described species Streptomyces qinglanensis DSM 42035T. The average nucleotide identity value between strain TRM 66187T and the closest related strain Streptomyces qinglanensis DSM 42035T was calculated to be 77.2%. The digital DNA-DNA hybridization value between them was 22.4%. Multilocus sequence analyses based on five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) also indicated that strain TRM 66187T should be assigned to the genus Streptomyces. On the basis of evidence from this polyphasic study, strain TRM 66187T should be designated as representing a novel species of the genus Streptomyces, for which the name Streptomyces lycii sp. nov. is proposed. The type strain is TRM 66187T (=LMG 31493T=CCTCC AA 2018094T).

Determination of five Alternaria toxins in wolfberry using modified QuEChERS and ultra-high performance liquid chromatography-tandem mass spectrometry.

A modified quick, easy, cheap, effective, rugged, and safe method coupled with ultra-high performance liquid chromatography-tandem mass spectrometry was established for the simultaneous detection of five Alternaria toxins (tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene, and tentoxin) in wolfberry. The sample pretreatment conditions including the dilution solvent, the extraction solvent and the QuEChERS purification parameters were optimized. Detection of the five Alternaria toxins was performed in MRM mode under ESI + conditions. The results showed that the five Alternaria toxins exhibitedgood linearity (1-200 µg/L, with correlation coefficients > 0.999). The limits of detection were 0.07-0.24 µg/kg, and the limits of quantification were 0.32-0.77 µg/kg. The trueness was between 73.8% and 111.5%, and the precision was lower than 10%. The established method was effectively used for the determination of five Alternaria toxins in 155 wolfberry samples from Northwest China.

Evaluation of the rust fungus Puccinia rapipes for biological control of Lycium ferocissimum (African boxthorn) in Australia: Life cycle, taxonomy and pathogenicity.

Fungal plant pathogens are increasingly recognised as being among the most effective and safe agents in classical weed biological control programs worldwide. Suitability of the rust fungus P. rapipes as a classical biological control agent for Lycium ferocissimum (African boxthorn) in Australia was assessed using a streamlined agent selection framework. Studies with P. rapipes were undertaken to elucidate its life cycle, confirm its taxonomic placement and determine its pathogenicity to L. ferocissimum and seven closely-related Solanaceae species that occur in Australia. Field surveys in the native range of South Africa, experiments in a containment facility in Australia and DNA sequencing confirmed that P. rapipes is macrocyclic and autoecious, producing all five spore stages on L. ferocissimum. The stages not previously encountered, spermogonia and aecia, are described. Sequencing also confirmed that P. rapipes is sister to Puccinia afra, in the 'Old World Lineage' of Puccinia species on Lycieae. Two purified isolates of the fungus, representing the Eastern and Western Cape distributions of P. rapipes in South Africa, were cultured in the containment facility for use in pathogenicity testing. L. ferocissimum and all of the Lycium species of Eurasian origin tested ‒ Lycium barbarum (goji berry), Lycium chinense (goji berry 'chinense') and Lycium ruthenicum (black goji berry) - were susceptible to both isolates of P. rapipes. The Australian native L. australe and three more distantly related species in different genera tested were resistant to both isolates. The isolate from the Western Cape was significantly more pathogenic on L. ferocissimum from Australia, than the Eastern Cape isolate. Our results indicate that P. rapipes may be sufficiently host specific to pursue as a biological control agent in an Australian context, should regulators be willing to accept damage to the Eurasian goji berries being grown, albeit to a limited extent, in Australia.

Absolute Configuration and Biological Activities of Meroterpenoids from an Endophytic Fungus of Lycium barbarum.

Eleven new meroterpenoids, bipolahydroquinones A-C (1-3), cochlioquinones I-N (4-8 and 11), and isocochlioquinones F and G (9 and 10), together with six known ones, were obtained from an endophytic fungus Bipolaris sp. L1-2 from Lycium barbarum. Compound 2 is the first example of cochlioquinone derivatives possessing a benzofuran moiety. The structures and absolute configurations of the new compounds were elucidated by spectroscopic data interpretation, single-crystal X-ray diffraction analysis, chemical transformations, and biosynthetic considerations. Compounds 3, 4, and 6-8 showed cytotoxicity against NCI-H226 and/or MDA-MB-231 with IC50 values ranging from 5.5 to 9.5 µM.

Complete sequence of a novel alphaendornavirus from the phytopathogenic fungus Arthrocladiella mougeotii.

A novel double-stranded RNA (dsRNA) virus, which was provisionally named Arthrocladiella mougeotii endornavirus (AmEV), was isolated from Arthrocladiella mougeotii, the phytopathogenic fungus infecting Lycium chinense in Beijing, China. The genome of AmEV is 11,683 nucleotides in length with a 5' and 3' non-coding region of 16 and 50 nt, respectively, as well as a single 11,617-nt long open reading frame potentially encoding a putative protein of 3,871 amino acids with conserved Helicase and RNA-dependent RNA polymerase (RdRp) domains. Phylogenetic analysis based on the the amino acid sequence of the RdRp showed that AmEV is most closely related to Erysiphe cichoracearum endornavirus (EcEV).

Nocardia rhizosphaerihabitans sp. nov., a novel actinomycete isolated from a coastal soil.

An actinomycete strain, designated KLBMP S0039T, was isolated from the rhizosphere soil of Lycium Linn., collected from the coastal region in Lianyungang, Jiangsu Province, eastern PR China, and was studied to determine its taxonomic position. The isolate showed a combination of morphological and chemotaxonomic properties typical of the members of the genus Nocardia. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were galactose, arabinose, glucose and ribose. The predominant menaquinone was identified as MK-8(H4ω-cycl). The diagnostic phospholipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and unknown lipids. The predominant cellular fatty acids were identified as C16 : 0, C18 : 0, C18 : 1ω9c, and 10-methyl C18 : 0 [tuberculostearic acid (TBSA)]. The G+C content of the genomic DNA was determined to be 68.2 mol%. The 16S rRNA gene sequence similarity indicated that KLBMP S0039T was most closely related to Nocardia neocaledoniensis NBRC 108232T (99.4 % 16S rRNA gene sequence similarity) and Nocardia asteroides NBRC 15531T (99.2 %), similarities to other type strains of species of the genus Nocardia were found to be less than 98.6 %. However, DNA-DNA relatedness values and phenotypic data indicated that KLBMP S0039T could be clearly distinguished from the closely related species of the genus Nocardia. On the basis of polyphasic taxonomic data, it is concluded that KLBMP S0039T represents a novel species of the genus Nocardia, for which the name Nocardiarhizosphaerihabitans sp. nov. is proposed. The type strain is KLBMP S0039T (=KCTC 39693T=CGMCC 4.7329T).

Plant-plant competition outcomes are modulated by plant effects on the soil bacterial community.

Competition is a key process that determines plant community structure and dynamics, often mediated by nutrients and water availability. However, the role of soil microorganisms on plant competition, and the links between above- and belowground processes, are not well understood. Here we show that the effects of interspecific plant competition on plant performance are mediated by feedbacks between plants and soil bacterial communities. Each plant species selects a singular community of soil microorganisms in its rhizosphere with a specific species composition, abundance and activity. When two plant species interact, the resulting soil bacterial community matches that of the most competitive plant species, suggesting strong competitive interactions between soil bacterial communities as well. We propose a novel mechanism by which changes in belowground bacterial communities promoted by the most competitive plant species influence plant performance and competition outcome. These findings emphasise the strong links between plant and soil communities, paving the way to a better understanding of plant community dynamics and the effects of soil bacterial communities on ecosystem functioning and services.

[Antimicrobial and anti-tumor activities of endophytes from Lycium barbarum of Ningxia].

The antimicrobial activity and cytotoxicity in vitro of the fermentation broth of 10 endophytic strains isolated from Lycium barbarum were determined to screen high activity endophytic strains. Sequences analysis of ITS and 16S rDNA was used for molecular identification of the strains. The results showed that 5 endophytic fungi had no inhibitory activity against the tested pathogens. Endophytic actinomycete strain AL6 had a certain inhibitory effect on 3 kinds of pathogenic fungi, and strain AL5 only had strong inhibitory activity against Staphylococcus aureus. However, the anti-tumor activity of endophytic fungi was significantly higher than that of actinomycetes. Four endophytic fungi strains exhibited the growth inhibition rate of above 50% against at least one of the tested tumor cells when the concentration of fermentation broth was 0.2 g•L⁻¹. Sequences analysis showed that 5 endophytic fungi strains belonged to genus Aspergillus, Penicillium and Emericella, and the 5 endophytic actinomycetes strains belonged to genus Aspergillus, Penicillium and Emericella. Aspergillus strain FL1 had stronger inhibitory activity against A549 and HeLa cells, and the IC50 values were 0.022，0.028 g•L⁻¹, respectively, which was worthy of further study.

Arbuscular mycorrhizas influence Lycium barbarum tolerance of water stress in a hot environment.

Arbuscular mycorrhizal (AM) fungi can assist their hosts to cope with water stress and other abiotic stresses in different ways. In order to test whether AM plants have a greater capacity than control plants to cope with water stress, we investigated the water status and photosynthetic capacity of Lycium barbarum colonized or not by the AM fungus Rhizophagus irregularis under three water conditions during a hot summer. Sugar levels and transcriptional responses of both plant and AM fungus aquaporin genes in roots were analyzed. Compared with control plants, AM plants increased transpiration rate and stomatal conductance but decreased leaf relative water content under moderate water stress. Severe water stress, however, did not inhibit the quantum yield of PSII photochemistry in AM plants versus control plants. AM plants had higher expression levels of plasma membrane intrinsic proteins or tonoplast intrinsic proteins and Rir-AQP2 and lower leaf temperature than control plants under dry-hot stress. Additionally, AM plant sugar levels under normal water conditions were similar to those of control plants under moderate water stress, but sugar levels of AM plants especially increased with severe water stress. When these aspects of performance of AM and control plants under different water conditions are compared overall, AM plants displayed an obvious superiority over control plants at coping with moderate water stress in the hot environment; AM plants maintained normal photochemical processes under severe water stress, while sugar levels were affected strongly.

Influence of Rhizoglomus irregulare on nutraceutical quality and regeneration of Lycium barbarum leaves under salt stress.

Whether arbuscular mycorrhizal fungi augment the nutraceutical quality of crops under salt stress is critical as a potential agronomic practice in salinized farmland. To evaluate the effect of Rhizoglomus irregulare on the nutraceutical quality of Lycium barbarum leaves under salt stress, we analyzed growth parameters and the rutin, polysaccharide, acidic polysaccharide, and amino acids contents of 2 harvests. Inoculation of R. irregulare significantly increased the regenerated bud number (partial eta squared (PES) = 0.577, P < 0.0001) and rutin concentration (PES = 0.544, P < 0.001) of L. barbarum leaves, with and without salt stress. The biomass of the 2nd harvest (PES = 0.355, P = 0.0091) and acidic polysaccharide (PES = 0.518, P = 0.001) of L. barbarum leaves were notably enhanced by R. irregulare under 200 mmol/L salt level. Rhizoglomus irregulare had insignificant effect on polysaccharide (PES = 0.092, P = 0.221) and amino acids levels (PES = 0.263, P = 0.130) in the leaves of L. barbarum. However, inoculation by R. irregulare decreased proline level (PES = 0.761, P = 0.001) in the leaves of L. barbarum when subjected to salt stress. Taken together, these results indicate that R. irregulare significantly improved the nutraceutical quality and facilitated the sustainable production of L. barbarum leaves exposed to salt stress.

Pichia kudriavzevii as a representative yeast of North Patagonian winemaking terroir.

Terroir concept includes specific soil, topography, climate, landscape characteristics and biodiversity features. In reference to the last aspect, recent studies investigating the microbial biogeography (lately called 'microbial terroir') have revealed that different wine-growing regions maintain different microbial communities. The aim of the present work was to identify potential autochthonous fermentative yeasts isolated from native plants in North Patagonia, Schinus johnstonii, Ephedra ochreata and Lycium chilense, that could be associated to the specific vitivinicultural terroir of this region. Different Pichia kudriavzevii isolates were recovered from these plants and physiologically and genetically compared to regional wine isolates and foreign reference strains of the same species. All isolates were subjected to molecular characterization including mtDNA-RFLP, RAPD-PCR and sequence analysis. Both wine and native P. kudriavzevii isolates from Patagonia showed similar features, different from those showed by foreign strains, suggesting that this species could be part of a specific regional terroir from North Patagonia.

Micromonospora lycii sp. nov., a novel endophytic actinomycete isolated from wolfberry root (Lycium chinense Mill).

A novel actinomycete, designated strain NEAU-gq11(T), was isolated from wolfberry root (Lycium chinense Mill) and characterized using a polyphasic approach. Strain NEAU-gq11(T) was observed to form rough-surfaced spores that borne singly on the substrate hyphae but aerial mycelia were not developed. The organism showed closest 16S rRNA gene sequence similarity to Micromonospora saelicesensis Lupac 09(T) (99.4%), and phylogenetically clustered with M. chokoriensis 2-19/6(T) (99.3%), 'M. zeae' NEAU-gq9(T) (99.3%), M. violae NEAU-zh8(T) (99.1%), 'M. jinlongensis' NEAU-GRX11(T) (99.1%), M. zamorensis CR38(T) (99.0%), M. taraxaci NEAU-P5(T) (98.9%) and M. lupini Lupac 14N(T) (98.6%). Phylogenetic analysis based on the gyrB gene sequences also indicated that the isolate clustered with the above type strains except M. violae NEAU-zh8(T). The cell-wall peptidoglycan consisted of meso-diaminopimelic acid and glycine. The major menaquinones were MK-9(H8), MK-10(H6) and MK-10(H2). The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acid was iso-C16:0. Furthermore, some physiological and biochemical properties and low DNA-DNA relatedness values enabled the strain to be differentiated from members of closely related species. Therefore, it is proposed that strain NEAU-gq11(T) represents a novel species of the genus Micromonospora, for which the name Micromonospora lycii sp. nov. is proposed. The type strain is NEAU-gq11(T) (=CGMCC 4.7149(T)=DSM 46703(T)).

A new pyrone derivative from an endophytic Aspergillus tubingensis of Lycium ruthenicum.

A new pyrone named 6-isovaleryl-4-methoxy-pyran-2-one (1), along with three known pyrone compounds, rubrofusarin B (2), asperpyrones A (3) and campyrone A (4), was isolated from fermentation of Aspergillus tubingensis in Lycium ruthenicum. Their structures were confirmed by spectroscopic techniques, such as IR, NMR and HRESI-MS. Compound 2 indicated strong inhibitory activity against Escherichia coli, with MIC value of 1.95 µg/mL.

[Screening and identification of antioxidant endophytes from Lycium barbarum of Ningxia].

In this paper, 29 endophytes were isolated from different organs and tissues of Lycium barbarum of Ningxia by tablet coating method, 18 of them was fungi, and 11 of them was actinomycetes. The endophytes quantity in the different tissues were leaves > flowers > roots >fruits; The hydroxyl radical scavenging activities of 11 endophytes were investigated by Fenton reaction, and total antioxidant capacities of them were examined by a. total antioxidant capacity test kit; culture features and strain-specific sequence analysis were employed to explore the diversity of the 11 endophytes. The result showed that 5 fungi and 6 actinomycetes that having antioxidant activity could be phylogenetically classified into 3 genera, 3 genera and 3 families, respectively. The total antioxidant capacity and hydroxyl radical scavenging activity of the 11 endophytes showed distinct difference. The antioxidant activity of Aspergillus were stronger, among which total antioxidant capacity of fL1 was (188.5 ± 0.549) U · mL⁻¹ and the IC50 was 0.3 mg · L⁻¹; the IC50 of strain fL1 was 0.42 mg · L⁻¹ and the total antioxidant capacity of fL9 was (113.63 ± 1.021) U · mL⁻¹, all of them were stronger than the positive control Vit C. The experimental results indicated that endophytic fungi of L. barbarum of Ningxia have a great developing and application prospect for the development of antioxidant agent.

[Investigation of production status in major wolfberry producing areas of China and some suggestions].

To investigate the production status and the safety influence factors of wolfberry in China. We investigated the detailed factors which affect the quality safe of wolfberry in the periods of July-August 2013 and July-September 2009. The factors include fertilizing patterns, the used pesticide and preliminary process wolfberry. The factors were discussed according to the results of investigation, and suggestions were proposed for the management and production departments of wolfberry.

Endophytic fungi from Lycium chinense Mill and characterization of two new Korean records of Colletotrichum.

Chinese boxthorn or matrimony vine (Lycium chinense Mill) is found primarily in southeastern Europe and Asia, including Korea. The dried ripe fruits are commonly used as oriental medicinal purposes. Endophytic fungi were isolated from surface sterilized tissues and fruits of the medicinal plant in 2013 to identify the new or unreported species in Korea. Among 14 isolates, 10 morphospecies were selected for molecular identification with the internal transcribed spacer (ITS) gene. Phylogenetic analysis revealed that all isolates belonged to Ascomycota including the genera Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea and Nemania. Two Colletotrichum species were identified at the species level, using three genes including internal transcribed spacer (ITS), glycerol-3-phosphate dehydrogenase (GAPDH) and Actin (ACT) for PCR and molecular data analysis along with morphological observations. The fungal isolates, CNU122031 and CNU122032 were identified as Colletotrichum fructicola and C. brevisporum, respectively. Morphological observations also well supported the molecular identification. C. brevisporum is represented unrecorded species in Korea and C. fructicola is the first record from the host plant.