The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses.

BACKGROUND: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear. RESULTS: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity. CONCLUSIONS: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing the transcription factor ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

Debaryomyces nepalensis reduces fungal decay by affecting the postharvest microbiome during jujube storage.

Microbial antagonists are effective and environmentally friendly in controlling postharvest diseases of fruit. The present study investigated the influence of D. nepalensis on epiphytic microbiome and postharvest decay of jujube. Results showed that D. nepalensis notably reduced fungal decay, maintained the fruit firmness and delayed discoloration. The epiphytic microbiome revealed that D. nepalensis changed the fungal communities, but few influence on bacterial communities were observed. D. nepalensis, as the dominant population in the treatment group, decreased the abundance of pathogenic fungi of Alternaria, Penicillium, Fusarium and Botrytis, while increased the beneficial bacteria of Pantoea. The canonical correspondence analysis revealed that Debaryomyces was negatively correlated with the decay rate, whereas Penicillium, Acremonium, Rhodosporidiobolus and Hansfordia were positively correlated. In conclusion, D. nepalensis altered the successional process of fungal and bacterial communities to reduce the decay rate of jujube during storage.

Ozone reduces the fruit decay of postharvest winter jujube by altering the microbial community structure on fruit surface.

Microbial community structure on fruit surface plays an important role in fruit decay during postharvest storage, although the underlying mechanism has not been fully elucidated. Winter jujube (Ziziphus jujuba Miller cv. Dongzao) is a unique fruit resource with high edible and commercial value in China, while postharvest decay has always been a severe problem leading to short shelf life and poor quality of fruit. Ozone treatment is regarded as one of the most effective means to control decay and extend shelf life because of its cost-effective and eco-friendly properties. In the present study, three concentrations of ozone (2.5, 5 and 10 µL L-1) were found to reduce significantly postharvest decay of winter jujube on days 10 and 15, which were produced from Huanghua City, Hebei, China. High-throughput sequencing revealed significant changes in the bacterial and fungal communities in response to the application of ozone treatment, while Didymella, Rhizopus, Alternaria, Phialemoniopsis and Mycosphaerella were found to be the most abundant in fungi, and Methylobacterium, Pseudomonas, Pantoea, Sphingomonas and Gluconobacter being the most abundant in bacteria. Results of linear discriminant analysis (LDA) effect size (LEfSe) indicated that ozone treatments considerably reduced the abundance of Rhizopus and Gluconobacter on the surface of winter jujube fruit. Furthermore, Pearson correlation analysis showed that Rhizopus was positively correlated with Gluconobacter (r = 0.97) while negatively correlated with Didymella (r = -0.96). By predicting the metabolic function, ozone may inhibit metabolic pathways including nucleoside and nucleotide biosynthesis, amino acid biosynthesis, fatty acid and lipid degradation, respiration, and electron transfer, thereby reducing the incidence of fruit decay and maintaining the firmness of winter jujube fruit.

The Effect of Salt-Tolerant Antagonistic Bacteria CZ-6 on the Rhizosphere Microbial Community of Winter Jujube (Ziziphus jujuba Mill. "Dongzao") in Saline-Alkali Land.

As the main economic crop cultivated in the Yellow River Delta, winter jujube contains various nutrients. However, soil salinization and fungal diseases have affected the yield and quality of winter jujube. In order to use plant growth-promoting rhizobacteria (PGPR) to reduce these damages, the antagonistic bacteria CZ-6 isolated from the rhizosphere of wheat in saline soil was selected for experiment. Gene sequencing analysis identified CZ-6 as Bacillus amyloliquefaciens. In order to understand the salt tolerant and disease-resistant effects of CZ-6 strain, determination of related indicators of salt tolerance, pathogen antagonistic tests, and anti-fungal mechanism analyses was carried out. A pot experiment was conducted to evaluate the effect of CZ-6 inoculation on the rhizosphere microbial community of winter jujube. The salt tolerance test showed that CZ-6 strain can survive in a medium with a NaCl concentration of 10% and produces indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Studies on the inhibition mechanism of pathogenic fungi show that CZ-6 can secrete cellulase, protease, and xylanase. Gas chromatography-mass spectrometry (GC-MS) analysis showed that CZ-6 can release volatile organic compounds (VOCs), including 2-heptanone and 2-nonanone. In addition, the strain can colonize the rhizosphere and migrate to the roots, stems, and leaves of winter jujube, which is essential for plant growth or defense against pathogens. Illumina MiSeq sequencing data indicated that, compared to the control, the abundance of salt-tolerant bacteria Tausonia in the CZ-6 strain treatment group was significantly increased, while the richness of Chaetomium and Gibberella pathogens was significantly reduced. Our research shows that CZ-6 has the potential as a biological control agent in saline soil. Plant damage and economic losses caused by pathogenic fungi and salt stress are expected to be alleviated by the addition of salt-tolerant antagonistic bacteria.

Epsilon-poly-l-lysine (ε-PL) exhibits multifaceted antifungal mechanisms of action that control postharvest Alternaria rot.

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial poly-cationic peptide widely applied as a natural preservative in the food industry, whereas its application in preventing postharvest loss of fruit was largely absent. This study investigated the antifungal activity of ε-PL and determined the possible mechanisms involved. The in vivo results indicated that 500 mg L-1 exogenous ε-PL treatment significantly inhibited black spot rot in apple, jujube, and tomato. The lesion diameter inhibition rate was range from 20.11% to 29.09% by 500 mg L-1 ε-PL treatment. ε-PL exerts antifungal activity against A. alternata in vitro, the half-inhibition concentration is 160.1 mg L-1. ε-PL induced morphology and ultrastructure change on the pathogen, which resulted in the inhibition of A. alternata. This was accomplished by disturbing pathogen membrane integrity and functionality. The fluorometric assay confirmed that ε-PL induced endogenous reactive oxygen species formation and accumulation in A. alternata and the elicited severe lipid peroxidation that caused membrane lesions. Further, ε-PL treatment enhanced the expression of genes involved in antioxidant metabolism and pathogenesis-related responses in apple fruit. These findings illustrated that ε-PL exhibits multifaceted antifungal activity by the direct effect on the pathogen as well as induce host defense responses. ε-PL may be conducive as a promising alternative for Alternaria rot management.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Molecular characterization of a novel partitivirus isolated from the phytopathogenic fungus Aplosporella javeedii.

Aplosporella javeedii is a pathogenic fungus that causes canker and dieback of jujube in China. In this study, we report a new mycovirus, Aplosporella javeedii partitivirus 1 (AjPV1), isolated from A. javeedii strain NX55-3. The AjPV1 genome contains two double-stranded RNA elements (dsRNA1 and dsRNA2). The size of dsRNA1 is 2,360 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp), while dsRNA2 is 2,301 bp in length and encodes a putative capsid protein (CP). The sequences of RdRp and CP have significant similarity to those of members of the family Partitiviridae. Sequence alignment and phylogenetic analysis showed that AjPV1 is a new member of the family Partitiviridae that is related to members of the genus Betapartitivirus. To our knowledge, AjPV1 is the first mycovirus reported from A. javeedii.

Isolation and characterization of endophytic bacteria for controlling root rot disease of Chinese jujube.

AIM: Fusarium oxysporum is the primary pathogen causing root rot disease that severely affects cultivation of jujube fruit in the Xinjiang province of China. The aim of this study was to identify endophytic bacteria in healthy jujube organs that could effectively suppress F. oxysporum growth. METHODS AND RESULTS: Different plant organs (leaves, twigs and roots) were collected from healthy Chinese jujube cultivated in southern Xinjiang province of China. The endophytic bacterium Brevibacterium halotolerans JZ7 was selected for its strong antagonistic activity and growth-promoting characteristics. Gas-chromatography mass-spectrometry analysis showed that acetoin, 2,3-butanediol and fenretinide were the three dominant volatile organic compounds produced by strain JZ7. Fenretinide strongly suppressed spore germination of F. oxysporum in vitro. Pot experiments showed that strain JZ7 colonized both the roots and rhizosphere soil of Chinese jujube and significantly reduced F. oxysporum level in jujube rhizosphere soil. CONCLUSION: We demonstrated that B. halotolerans JZ7 can be developed into a biological control agent to combat root rot disease of Chinese jujube in the Xinjiang province of China. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested strategy for biological control of jujube root rot disease is fully in accordance with the current principles of sustainability.

Genome-wide identification of MAPKKK genes and their responses to phytoplasma infection in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Mitogen-activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to a wide range of biotic and abiotic stresses. In a previous study, we identified ten ZjMAPKs and five ZjMAPKKs in the Chinese jujube genome. We found that some members of ZjMAPKs and ZjMAPKKs may play key roles in the plant's response to phytoplasma infection. However, how these ZjMAPKKs are modulated by ZjMAPKKKs during the response process has not been elucidated. Little information is available regarding MAPKKKs in Chinese jujube. RESULTS: A total of 56 ZjMAPKKKs were identified in the jujube genome. All of these kinases contain the key S-TKc (serine/threonine protein kinase) domain, which is distributed among all 12 chromosomes. Phylogenetic analyses show that these ZjMAPKKKs can be classified into two subfamilies. Specifically, 41 ZjMAPKKKs belong to the Raf subfamily, and 15 belong to the MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures. Only one pair of ZjMAPKKKs (15/16, on chromosome 5) was found to be tandemly duplicated. Using qPCR, the expression profiles of these MAPKKKs were investigated in response to infection with phytoplasma. In the three main infected tissues (witches' broom leaves, phyllody leaves, and apparently normal leaves), ZjMAPKKK26 and - 45 were significantly upregulated, and ZjMAPKKK3, - 43 and - 50 were significantly downregulated. ZjMAPKKK4, - 10, - 25 and - 44 were significantly and highly induced in sterile cultivated tissues infected by phytoplasma, while ZjMAPKKK6, - 7, - 17, - 18, - 30, - 34, - 35, - 37, - 40, - 41, - 43, - 46, - 52 and - 53 were significantly downregulated. CONCLUSIONS: For the first time, we present an identification and classification analysis of ZjMAPKKKs. Some ZjMAPKKK genes may play key roles in the response to phytoplasma infection. This study provides an initial understanding of the mechanisms through which ZjMAPKKKs are involved in the response of Chinese jujube to phytoplasma infection.

[Investigation of aflatoxins,mycobiota,and toxigenic fungi during post-harvest handling of Ziziphi Spinosae Semen].

Ziziphi Spinosae Semen is one of the Chinese herbal medicine being susceptible to aflatoxins contamination. To investigate the sources of aflatoxins contamination and toxigenic fungi species on Ziziphi Spinosae Semen,32 samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxins in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. The dilution-plate method was applied to the fungi isolation. The isolated fungi strains were identified by morphological characterization and molecular approaches. The results showed that aflatoxins were detected in 28 samples from every step during the processing of Ziziphi Spinosae Semen. Three samples were detected with aflatoxin B_1 and 2 samples with both aflatoxin B_1 and total aflatoxin exceeding the limit of Chinese Pharmacopoeia. Especially the samples from the washing step,with the highest detected amounts of AFB_1 and AFs were reached 94. 79,121. 43 µg·kg~(-1),respectively. All 32 samples were contaminated by fungi. The fungal counts on the newly harvested samples were 2. 20 × 10~2 CFU·g~(-1). Moreover,it increased as tphreocessing progresses,and achieved 1. 16×10~6 CFU·g~(-1) after washing. A total of 321 isolates were identified to 17 genera. Aspergillus flavus was the main source of aflatoxins during the processing and storage of Ziziphi Spinosae Semen. One isolate of A. flavus was confirmed producing AFB_1 and AFB_2. The fungal count was significantly increased by composting,and Aspergillus was the predominant genus after shell breaking. The contamination level of aflatoxins was increased by composting and washing.

Genome-wide analysis of the bHLH gene family in Chinese jujube (Ziziphus jujuba Mill.) and wild jujube.

BACKGROUND: The bHLH (basic helix-loop-helix) transcription factor is one of the largest families of transcription factors in plants, containing a large number of members with diverse functions. Chinese jujube (Ziziphus jujuba Mill.) is the species with the highest economic value in the family Rhamnaceae. However, the characteristics of the bHLH family in the jujube genome are still unclear. Hence, ZjbHLHs were first searched at a genome-wide level, their expression levels under various conditions were investigated systematically, and their protein-protein interaction networks were predicted. RESULTS: We identified 92 ZjbHLHs in the jujube genome, and these genes were classified into 16 classes according to bHLH domains. Ten ZjbHLHs with atypical bHLH domains were found. Seventy ZjbHLHs were mapped to but not evenly distributed on 12 pseudo- chromosomes. The domain sequences among ZjbHLHs were highly conserved, and their conserved residues were also identified. The tissue-specific expression of 37 ZjbHLH genes in jujube and wild jujube showed diverse patterns, revealing that these genes likely perform multiple functions. Many ZjbHLH genes were screened and found to be involved in flower and fruit development, especially in earlier developmental stages. A few genes responsive to phytoplasma invasion were also verified. Based on protein-protein interaction prediction and homology comparison, protein-protein interaction networks composed of 92 ZjbHLHs were also established. CONCLUSIONS: This study provides a comprehensive bioinformatics analysis of 92 identified ZjbHLH genes. We explored their expression patterns in various tissues, the flowering process, and fruit ripening and under phytoplasma stress. The protein-protein interaction networks of ZjbHLHs provide valuable clues toward further studies of their biological functions.

Genome-wide analysis of the WRKY gene family and their positive responses to phytoplasma invasion in Chinese jujube.

BACKGROUND: The WRKY gene family is one of the most important families in higher plants. As transcription factors, they actively respond to biotic and abiotic stress and are also involved in plant development. Chinese jujube (Ziziphus jujuba Mill.) is the largest type of dried fruit tree in China in terms of production, but its production is largely limited by phytoplasma infection, and the information about the role of WRKY genes under phytoplasma stress was still limited. RESULTS: We identified 54 ZjWRKYs in the jujube genome and classified them into three subgroups according to conserved WRKY domains and zinc-finger structure. 41 ZjWRKYs were distributed on 11 of 12 pseudo chromosomes in Chinese jujube. The majority of ZjWRKYs were highly expressed in the seven examined tissues, indicating that they play multiple roles in these vegetative and reproductive organs. Transcriptome data showed that most of the characterised ZjWRKYs were highly expressed at later stages of fruit development. RT-qPCR demonstrated that the expression of 23 ZjWRKYs changed following phytoplasma infection, suggesting that they are involved in signalling pathways that respond to phytoplasma stress. Then, STRING analysis and yeast two-hybrid screening proved that some ZjWRKY proteins were interacting with ZjMAPKK proteins, which were also involved in phytoplasma invasion. Moreover, their differential expressions were further confirmed in resistant and susceptible jujube varieties under phytoplasma stress. These results suggest that ZjWRKYs play significant roles in phytoplasma tolerance and should be crucial candidate genes for jujube-phytoplasma interaction. CONCLUSIONS: 54 ZjWRKYs in Chinese jujube were identified and classified into three subgroups. 41 ZjWRKYs were unevenly distributed along the chromosomes. The majority of ZjWRKYs were highly expressed in various tissues. Most of the ZjWRKYs were positive responses to phytoplasma invasion, and that provided candidate genes for the future studies of jujube-phytoplasma interaction.

Defense Responses, Induced by p-Coumaric Acid and Methyl p-Coumarate, of Jujube ( Ziziphus jujuba Mill.) Fruit against Black Spot Rot Caused by Alternaria alternata.

The esterified fraction of jujube ( Ziziphus jujuba Mill.) peel extract showed strong antifungal activity on Alternaria alternata. p-Coumaric acid (pCA) was found to be the most predominant individual phenolic acid that was correlated highly with the antifungal activity of the esterified fraction. Thus, effects of postharvest treatments with pCA and its simplest esterified derivative methyl p-coumarate (MeCA) against black spot rot on jujube fruit caused by A. alternata were investigated. pCA and MeCA strongly suppressed in vitro growth of the fungus and significantly reduced postharvest Alternaria rot on fresh jujubes. Biochemical and transcriptional analysis revealed that pCA and MeCA regulated the expression of some genes encoding antioxidant enzymes and their enzymatic activities, enhanced the phenylpropanoid pathway metabolism, and activated the expression of genes encoding pathogenesis-related proteins. These results suggested that, apart from its direct antifungal activity, pCA and MeCA induced defense responses in jujube fruit against postharvest Alternaria rot.

Rapid green synthesis of silver nano particles from Ziziphus mauritianaand antibacterial activity against human pathogens

A novel green source Ziziphus mauritiana fresh young leaves was opted to synthesize silver nanoparticles and analyze its antibacterial activity. The bioactive compounds present in the plant extracts reduced silver ions to NPs, indicated by change in colorfrom red to dark brown. In this study, we have successfully synthesized nanoparticles using Z.mauritiana aqueous leaf extract as a reducing agent and the reaction process of synthesized nanoparticles was monitored by UV-Vis spectroscopy. The UV-Vis absorption peak showed maximum adsorption at 420 nm confirmed the silver nanoparticles synthesis. Further characterization was carried out by FTIR and the results recorded a downward shift of absorption the bands between 400 to 4000 cm-1indicates the formationof silver nanoparticles. Finally, the present research was exploited to study the antibacterial activity of synthesized nanoparticles produced Z.mauritiana was studied using different pathogenic bacteria such as Salmonellasp., Proteus sp., Bacillus sp., Klebsiella pneumonia and E.colifrom the well diffusion results, the synthesized silver nanoparticles displayed the best antibacterial property as compared to the antibiotic has been reported in this paper. To the best of our knowledge, this is the first report that the Z.mauritiana aqueous extract facilitate the synthesis of silver nanoparticles and also exhibits antibacterial activity.

Tracing Rhizophagus irregularis isolate IR27 in Ziziphus mauritiana roots under field conditions.

Arbuscular mycorrhizal fungi (AMF) play a major role as biofertilizer for sustainable agriculture. Nevertheless, it is still poorly documented whether inoculated AMF can successfully establish in field soils as exotic AMF and improve plant growth and productivity. Further, the fate of an exogenous inoculum is still poorly understood. Here, we pre-inoculated two cultivars (Tasset and Gola) of the fruit tree Ziziphus mauritiana (jujube) with the exotic AM fungus Rhizophagus irregularis isolate IR27 before transplantation in the field. In two experiments, tracking and quantification of R. irregularis IR27 were assessed in a 13-month-old jujube and an 18-month-old jujube in two fields located in Senegal. Our results showed that the inoculant R. irregularis IR27 was quantitatively traced and discriminated from native R. irregularis isolates in roots by using a qPCR assay targeting a fragment of the RNA polymerase II gene (RPB1), and that the inoculum represented only fractions ranging from 11 to 15% of the Rhizophagus genus in the two plantations 13 and 18 months after transplantation, respectively. This study validates the use of the RPB1 gene as marker for a relative quantification of a mycorrhizal inoculant fungus isolate in the field.

Development and Application of a QuEChERS-Based Liquid Chromatography Tandem Mass Spectrometry Method to Quantitate Multi-Component Alternaria Toxins in Jujube.

A simple, rapid and efficient methodology was developed and validated for the analysis of four Alternaria toxins in jujube: Tenuazonic acid, alternariol, alternariol monomethyl ether, and tentoxin. Under the optimized extraction procedure, chromatographic conditions, and instrumental parameters, the four toxins were effectively extracted via a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, and quantified by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Matrix-matched calibrations ranging from 0.01 to 0.5 µg mL-1 were conducted for the quantification due to the matrix effect. A blank jujube sample was spiked at 40, 80 and 160 µg kg-1, obtaining recoveries in the range of 83.5⁻109.6%. Limits of detection and limits of quantification were in the range of 0.14⁻0.26 and 0.47⁻0.87 µg kg-1, respectively. Finally, the developed method was applied for the quantification of the four toxins in 14 jujube samples, including black spot-infected and uninfected samples. Results showed that the predominant toxin detected in all the samples was tenuazonic acid, the content of which was associated with the infection level; alternariol, alternariol monomethyl ether, and tentoxin were detected in all the infected samples and some of the uninfected samples with rather low contents.

Comparative genome analysis of jujube witches'-broom Phytoplasma, an obligate pathogen that causes jujube witches'-broom disease.

BACKGROUND: JWB phytoplasma is a kind of insect-transmitted and uncultivable bacterial plant pathogen causeing a destructive Jujube disease. To date, no genome information about JWB phytoplasma has been published, which hindered its characterization at genomic level. To understand its pathogenicity and ecology, the genome of a JWB phytoplasma isolate jwb-nky was sequenced and compared with other phytoplasmas enabled us to explore the mechanisms of genomic rearrangement. RESULTS: The complete genome sequence of JWB phytoplasma (jwb-nky) was determined, which consisting of one circular chromosome of 750,803 bp with a GC content of 23.3%. 694 protein-encoding genes, 2 operons for rRNA genes and 31 tRNA genes as well as 4 potential mobile units (PMUs) containing clusters of DNA repeats were identified. Based on PHIbaes analysis, a large number of genes were genome-specific and approximately 13% of JWB phytoplasma genes were predicted to be associated with virulence. Although transporters for maltose, dipeptides/oligopeptides, spermidine/putrescine, cobalt, Mn/Zn and methionine were identified, KEGG pathway analysis revealed the reduced metabolic capabilities of JWB phytoplasma. Comparative genome analyses between JWB phytoplasma and other phytoplasmas shows the occurrence of large-scale gene rearrangements. The low synteny with other phytoplasmas indicated that the expansion of multiple gene families/duplication probably occurred separately after differentiation. CONCLUSIONS: In this study, the complete genome sequence of a JWB phytoplasma isolate jwb-nky that causing JWB disease was reported for the first time and a number of species-specific genes were identified in the genome. The study enhanced our understandings about genomic basis and the pathogenicity mechanism of this pathogen, which will aid in the development of improved strategies for efficient management of JWB diseases.

Identification of endophytes with biocontrol potential from Ziziphus jujuba and its inhibition effects on Alternaria alternata, the pathogen of jujube shrunken-fruit disease.

Endophytic strains were isolated from different parts of a healthy "Dongzao" jujube (Ziziphus jujuba Mill. 'Dongzao') to find biocontrol agents against jujube shrunken-fruit disease caused by Alternaria alternata. The strains were screened using A. alternata strain CN193 as the target pathogen. The nutrient competition for all isolates was studied using the dual culture, and their inhibitive capability was tested by measuring the inhibition width of filter paper disks with filtrate. Influence of filtrate from the selected strains with strong inhibition of mycelial growth on spore germination was studied with hanging drop method on concavity slides. Colonization in the jujube leaves was assayed using a rifampicin-resistant mutant of strain St-zn-34 as the screening marker. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. A total of 81 endophytic strains were isolated from the stems, leaves, flowers, and fruits of winter jujube. Among these isolates, 14 strains showed strong antagonism against A. alternata. Further study showed that the filtrate of strains St-zn-9 and St-zn-34 could inhibit the mycelial growth of A. alternata, and the widths of their inhibition zone reached 6.14±0.03 mm and 8.27±0.09 mm, respectively. However, strain St-zn-34 showed stronger inhibition on spore germination than strain St-zn-9. St-zn-34 could significantly reduce the spore germination rate of A. alternata, and the spore did not germinate at all or the germ tube was very short. A rifampicin resistant-derivative of wild-type strain St-zn-34, which was designated as St-zn-34r, was obtained by transferring the strains to media with stepwise-increased rifampicin. Colonization assays indicated that St-zn-34r could colonize in jujube leaves, and the population of St-zn-34r was 1.2×103 CFU/g FW after inoculation for 30 days. Except for its salt tolerance, St-zn-34 was the closest to those of Bacillus subtilis. Thus, the strain was identified as B. subtilis.

Transcriptome profiling analysis revealed co-regulation of multiple pathways in jujube during infection by 'Candidatus Phytoplasma ziziphi'.

BACKGROUND: Jujube witches' broom (JWB), caused by a phytoplasma, devastates jujube tree (Ziziphus jujuba) growth and production in Asia. Although host responses to phytoplasmas are studied at the phenotypic, physiological, biochemical and molecular levels, it remains unclear how a host plant responds at the molecular level during the primary stage of infection. METHODS: To understand the response of the jujube tree to JWB infection, leaves were sampled at different times during the phytoplasma infection. Transcriptomic analyses at six stages were performed to reveal how phytoplasma infection affects Chinese jujube gene expression through the determination of the key differentially expressed genes (DEGs), and their related pathways. Quantitative real-time PCR was applied to validate 10 differentially expressed genes at different JWB phytoplasma infection stages. RESULTS: A total of 25,067 unigenes were mapped to jujube genome reference sequences. In the first infection stage (0-2 weeks after grafting (WAG), a total of 582 jujube genes were differentially regulated but no visible symptoms appeared. Quite a few DEGs related to abscisic acid (ABA) and cytokinin (CTK) were down-regulated, while some related to jasmonic acid (JA) and salicylic acid (SA) were up-regulated, Genes related to plant-pathogen interaction were also differentially expressed. In the second infection stage (37-39WAG), witches' broom symptoms were visible, and a total of 4373 DEGs were identified. Genes involved in biosynthesis and signal transduction of ABA, brassinosteroid (BR), CTK, ethylene (ET), and auxin (IAA), GA, JA and SA, plant-pathogen interaction, flavonoid biosynthesis genes were significantly regulated, suggesting that jujube trees activated defense factors related to SA, JA, ABA and secondary metabolites to defend against phytoplasma infection. By the third infection stage (48-52WAG), serious symptoms occurred and 3386 DEGs were identified. Most DEGs involved in biosynthesis and signal transduction of JA, SA and GA were up-regulated, while those relating to ABA were down-regulated. Genes involved in plant-pathogen interaction were up- or down-regulated, while phenylpropanoid and flavonoid biosynthesis genes were significantly up-regulated. Meanwhile, DEGs involved in photosynthesis, chlorophyll and peroxisome biosynthesis, and carbohydrate metabolism were down-regulated. These results suggested that phytoplasma infection had completely destroyed jujube trees' defense system and had disturbed chlorophyll synthesis and photosynthetic activity in the infected leaves at the late stage, resulting in yellow leaves and other JWB symptoms. DISCUSSION: The results in this report suggested that phytohormone biosynthesis and signal transduction, photosynthesis, and secondary metabolism all played important roles in the battle between colonization and defense in the interaction between Ca. Phytoplasma ziziphi and jujube.