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Sclerotinia disease is one of the most devastating fungal diseases worldwide, as it reduces the yields of many economically important crops. Pathogen-secreted effectors play crucial roles in infection processes. However, key effectors of Ciboria shiraiana, the pathogen primarily responsible for sclerotinia disease in mulberry (Morus spp.), remain poorly understood. In this study, we identified and functionally characterized the effector Cs02526 in C. shiraiana and found that Cs02526 could induce cell death in a variety of plants. Moreover, Cs02526-induced cell death was mediated by the central immune regulator brassinosteroid insensitive 1-associated receptor kinase 1, dependent on a 67-amino acid fragment. Notably, Cs02526 homologs were widely distributed in hemibiotrophic and necrotrophic phytopathogenic fungi, but the homologs failed to induce cell death in plants. Pretreatment of plants with recombinant Cs02526 protein enhanced resistance against both C. shiraiana and Sclerotinia sclerotiorum. Furthermore, the pathogenicity of C. shiraiana was diminished upon spraying plants with synthetic dsRNA-Cs02526. In conclusion, our findings highlight the cell death-inducing effector Cs02526 as a potential target for future biological control strategies against plant diseases., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Broussonetia papyrifera is widely found in cadmium (Cd) contaminated areas, with an inherent enhanced flavonoids metabolism and inhibited lignin biosynthesis, colonized by lots of symbiotic fungi, such as arbuscular mycorrhizal fungi (AMF). However, the physiological and molecular mechanisms by which Rhizophagus irregularis, an AM fungus, regulates flavonoids and lignin in B. papyrifera under Cd stress remain unclear. Here, a pot experiment of B. papyrifera inoculated and non-inoculated with R. irregularis under Cd stress was carried out. We determined flavonoids and lignin concentrations in B. papyrifera roots by LC-MS and GC-MS, respectively, and measured the transcriptional levels of flavonoids- or lignin-related genes in B. papyrifera roots, aiming to ascertain the key components of flavonoids or lignin, and key genes regulated by R. irregularis in response to Cd stress. Without R. irregularis, the concentrations of eriodictyol, quercetin and myricetin were significantly increased under Cd stress. The concentrations of eriodictyol and genistein were significantly increased by R. irregularis, while the concentration of rutin was significantly decreased. Total lignin and lignin monomer had no alteration under Cd stress or with R. irregularis inoculation. As for flavonoids- or lignin-related genes, 26 genes were co-regulated by Cd stress and R. irregularis. Among these genes, BpC4H2, BpCHS8 and BpCHI5 were strongly positively associated with eriodictyol, indicating that these three genes participate in eriodictyol biosynthesis and were involved in R. irregularis assisting B. papyrifera to cope with Cd stress. This lays a foundation for further research revealing molecular mechanisms by which R. irregularis regulates flavonoids synthesis to enhance tolerance of B. papyrifera to Cd stress., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene ( MnCYP710A11 ) was discovered in mulberry ( Morus notabilis ). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea , there was a notable increase in the expression of MnCYP710A11 . MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea . The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea , elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H 2 O 2 and O 2 - levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control., (© 2024 Scandinavian Plant Physiology Society.)

The genomes of obligately host-restricted bacteria suffer from accumulating mildly deleterious mutations, resulting in marked size reductions. Psyllids (Hemiptera) are phloem sap-sucking insects with a specialized organ called the bacteriome, which typically harbors two vertically transmitted bacterial symbionts: the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria) and a secondary symbiont that is phylogenetically diverse among psyllid lineages. The genomes of several Carsonella lineages were revealed to be markedly reduced (158-174‍ ‍kb), AT-rich (14.0-17.9% GC), and structurally conserved with similar gene inventories devoted to synthesizing essential amino acids that are scarce in the phloem sap. However, limited genomic information is currently available on secondary symbionts. Therefore, the present study investigated the genomes of the bacteriome-associated dual symbionts, Secondary_AM (Gammaproteobacteria) and Carsonella_AM, in the mulberry psyllid Anomoneura mori (Psyllidae). The results obtained revealed that the Secondary_AM genome is as small and AT-rich (229,822 bp, 17.3% GC) as those of Carsonella lineages, including Carsonella_AM (169,120 bp, 16.2% GC), implying that Secondary_AM is an evolutionarily ancient obligate mutualist, as is Carsonella. Phylogenomic ana-lyses showed that Secondary_AM is sister to "Candidatus Psyllophila symbiotica" of Cacopsylla spp. (Psyllidae), the genomes of which were recently reported (221-237‍ ‍kb, 17.3-18.6% GC). The Secondary_AM and Psyllophila genomes showed highly conserved synteny, sharing all genes for complementing the incomplete tryptophan biosynthetic pathway of Carsonella and those for synthesizing B vitamins. However, sulfur assimilation and carotenoid-synthesizing genes were only retained in Secondary_AM and Psyllophila, respectively, indicating ongoing gene silencing. Average nucleotide identity, gene ortholog similarity, genome-wide synteny, and substitution rates suggest that the Secondary_AM/Psyllophila genomes are more labile than Carsonella genomes.

A Pantoea ananatis strain, named LCFJ-001 (GDMCC: 1.6101), was isolated for the first time from bacterial wilt-diseased roots of mulberry ( Morus atropurpurea ) in the western part of the Guangxi Zhuang Autonomous Region, China. Moreover, through Koch's postulates, it was proven that LCFJ-001 can cause mulberry wilt, which is one of the pathogens of mulberry bacterial wilt. Here, we report a complete, annotated genome sequence of P. ananatis LCFJ-001. The entire genome sequence of P. ananatis strain LCFJ-001 was a 4,499,350 bp circular chromosome with 53.50% GC content. In total, 3,521 genes were annotated, of which 3,418 were assigned protein-coding genes. In addition, 22 ribosomal RNAs and 81 transfer RNAs were identified. The presented resource will help explore the pathogenetic mechanisms of mulberry wilt disease caused by the genus Pantoea ., Competing Interests: The author(s) declare no conflict of interest.

Endophytes have a wide range of potential in maintaining plant health and sustainable agricultural environmental conditions. In this study, we analysed the diversity of endophytic bacteria in four mulberry cultivars with different resistance capacity against bacterial wilt using metagenomic sequencing and culture-dependent approaches. We further assessed the role of 11 shared genera in the control of bacterial wilt of mulberry. The results of the present study showed that Actinobacteria, Firmicutes, and Proteobacteria were the three dominant phyla in all communities, with the representative genera Acinetobacter and Pseudomonas. The diversity analysis showed that the communities of the highly and moderately resistant varieties were more diverse compared to those of the weakly resistant and susceptible varieties. The control tests of mulberry bacterial wilt showed that Pantoea, Atlantibacter, Stenotrophomonas, and Acinetobacter were effective, with a control rate of over 80%. Microbacterium and Kosakonia were moderately effective, with a control rate between 50 and 80%. At the same time, Escherichia, Lysinibacillus, Pseudomonas, and Rhizobium were found to be less effective, with a control rate of less than 40%. In conclusion, this study provides a reasonable experimental reference data for the control of bacterial wilt of mulberry., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Drought stress often leads to heavy losses in mulberry planting, especially for fruits and leaves. Application of plant growth-promoting fungi (PGPF) endows various plant beneficial traits to overcome adverse environmental conditions, but little is known about the effects on mulberry under drought stress. In the present study, we isolated 64 fungi from well-growing mulberry trees surviving periodical drought stress, and Talaromyces sp. GS1, Pseudeurotium sp. GRs12, Penicillium sp. GR19, and Trichoderma sp. GR21 were screened out due to their strong potential in plant growth promotion. Co-cultivation assay revealed that PGPF stimulated mulberry growth, exhibiting increased biomass and length of stems and roots. Exogenous application of PGPF could alter fungal community structures in the rhizosphere soils, wherein Talaromyces was obviously enhanced after inoculation of Talaromyces sp. GS1, and Peziza was increased in the other treatments. Moreover, PGPF could promote iron and phosphorus absorption of mulberry as well. Additionally, the mixed suspensions of PGPF induced the production of catalase, soluble sugar, and chlorophyll, which in turn enhanced the drought tolerance of mulberry and accelerated their growth recovery after drought. Collectively, these findings might provide new insights into improving mulberry drought tolerance and further boosting mulberry fruit yields by exploiting interactions between hosts and PGPF.

The chronic nature of diabetes mellitus motivates the quest for novel agents to improve its management. The scarcity and prior uncontrolled utilization of medicinal plants have encouraged researchers to seek new sources of promising compounds. Recently, endophytes have presented as eco-friendly leading sources for bioactive metabolites. This article reviewed the endophytic fungi associated with Morus species and their isolated compounds, in addition to the biological activities tested on their extracts and chemical constituents. The relevant literature was collected from the years 2008-2022 from PubMed and Web of Science databases. Notably, no antidiabetic activity was reported for any of the Morus -associated endophytic fungal extracts or their twenty-one previously isolated compounds. This encouraged us to perform an in silico study on the previously isolated compounds to explore their possible antidiabetic potential. Furthermore, pharmacokinetic and dynamic stability studies were performed on these compounds. Upon molecular docking, Colletotrichalactone A ( 14 ) showed a promising antidiabetic activity due to the inhibition of the α-amylase local target and the human sodium-glucose cotransporter 2 (hSGT2) systemic target with safe pharmacokinetic features. These results provide an in silico interpretation of the possible anti-diabetic potential of Morus endophytic metabolites, yet further study is required.

Background: Feed shortage is a factor restricting animal production in the tropics, therefore how to use natural woody plant resources as animal feed is an important strategy., Results: Under the dual stress of an anaerobic and acidic environment, the microbial response during the fermentation of paper mulberry (PM) silage was found to be sensitive. The Gram-negative bacteria and mould died, and the dominant microbial community rapidly shifted to Gram-positive bacteria, resulting in a large reduction in microbial diversity and abundance. Exogenous bran additives interfered with the stress effects of the woody silage environment. Wheat bran (WB) accelerated the response of microorganisms to the anaerobic stress, and lactic acid bacteria became the dominant microbial community, thereby enhancing the lactic acid fermentation of silage, affecting the metabolic pathways of microorganisms, and improving the flavour and quality of the silage. Addition of rice bran made Enterobacter and Clostridium species quickly respond to the stress of the silage environment and become the predominant bacterial groups. In particular, anaerobic and spore-forming Clostridium species showed a strong tolerance to the silage environment, leading to butyric acid fermentation and protein degradation of the silage, and reducing its fermentation quality., Conclusion: The PacBio single-molecule real-time (SMRT) sequencing technology accurately revealed the microbial co-occurrence network and fermentation mechanism of silage. Our results indicate that PM can be used in combination with WB to prepare high-quality silage for animal production. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Chitinase is a hydrolase that uses chitin as a substrate. It plays an important role in plant resistance to fungal pathogens by degrading chitin. Here, we conducted bioinformatics analysis and transcriptome data analysis of the mulberry ( Morus notabilis ) chitinase gene family to determine its role in the resistance to Botrytis cinerea . A total of 26 chitinase genes were identified, belonging to the GH18 and GH19 families. Among them, six chitinase genes were differentially expressed under the infection of B. cinerea . MnChi18 , which significantly responded to B. cinerea , was heterologously expressed in Arabidopsis ( Arabidopsis thaliana ). The resistance of MnChi18 transgenic Arabidopsis to B. cinerea was significantly enhanced, and after inoculation with B. cinerea , the activity of catalase (CAT) increased and the content of malondialdehyde (MDA) decreased. This shows that overexpression of MnChi18 can protect cells from damage. In addition, our study also indicated that MnChi18 may be involved in B. cinerea resistance through other resistance-related genes. This study provides an important basis for further understanding the function of mulberry chitinase.

Objectives: Despite considerable advancement in antiretroviral therapy, development of safe, effective, and multi-targeted drugs for HIV still remains a big challenge. Endophytes are untouched and, hence, an important and novel sources in drug discovery endeavours. The present study was conducted to identify the anti-HIV compounds from Morus alba and endophytes isolated from it., Methods: The extracts of isolated endophytes were screened using high-performance liquid chromatography (HPLC). Further, all samples were analysed for their cytotoxicity using a thiazolyl blue tetrazolium bromide assay. Subsequently, anti-HIV activity was performed using cell-based and cell-free assay. At the end, potential endophytes were identified using gene sequencing., Results: A total of 27 endophytes were isolated from the eight stem bark samples of M. alba. Of the 27 endophytes, extracts of total of four endophytes showed a profile similar to the M. alba plant when analysed by HPLC. Further experimentation with extracts of these four endophytes, along with an extract of M. alba stem bark and its bioactive molecule, mulberroside C, revealed that all these six samples have good inhibitory potential for HIV. Among them, mulberroside C and two endophytic fungal extracts showed very potent anti-HIV activity. Subsequently, mechanistic studies at the molecular level showed that out of six test samples, three acted as protease inhibitors. Further, all four potential endophytes were identified using gene sequencing., Conclusions: The overall findings of these studies can help in the development of a novel anti-HIV candidate from mulberroside C, an extract of stem bark of M. alba and extracts of these endophytes. However, further validation and clinical studies are required to develop an anti-HIV drug., (© 2021 British HIV Association.)

Aims: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India., Methods and Results: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified., Conclusion: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India., Significance and Impact of the Study: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease., (© 2020 The Society for Applied Microbiology.)

The contribution of crops and soil microbial community structure and functional diversity in soil-borne diseases control mulberry plant production is still inadequately understood. In this work, a comparative study was undertaken on the microbial abundance, community structure, and functional diversity in the soil rhizosphere between the resistant (Kangqing 10) and the susceptible (Guisang 12) mulberry genotypes. The study deployed the use of dilution plate method, micro-ecology technology, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) techniques. The study aimed at developing better crop management methods for mulberry cultivation as well as preventing and controlling the occurrence and impacts of bacterial wilt on mulberry productivity. The results indicated that the soil rhizosphere microorganisms were more abundant in the normal resistant mulberry genotype than in the normal susceptible mulberry genotype. Carbon source utilization was better in the normal susceptible mulberry genotype. These properties were lower in the sickly resistant mulberry genotype than in the susceptible sickly mulberry genotype. Through the PCR-DGGE, it was shown that the bacterial and fungal community structures of the resistant genotypes were more stable than those of the susceptible genotypes. Through correlation regression analysis, it was shown that the mulberry bacterial wilt significantly contributes to the loss of soil nutrients, particularly organic matter and nitrogen, a possible cause to disrupted balance between the soil microbial community and the loss of soil organic matter. Resistant genotype plants displayed more resistance to bacterial wilt. Therefore, this study recommends the need to promote the cultivation of resistant genotype mulberry for increased yield.

Ciboria shiraiana causes hypertrophy sorosis scleroteniosis in mulberry trees, resulting in huge economic losses, and exploring its pathogenic mechanism at a genomic level is important for developing new control methods. Here, genome sequencing of C. shiraiana based on PacBio RSII and Illumina HiSeq 2500 platform as well as manual gap filling was performed. Synteny analysis with Sclerotinia sclerotiorum revealed 16 putative chromosomes corresponding to 16 chromosomes of C. shiraiana . Screening of rapid-evolution genes revealed that 97 and 2.4% of genes had undergone purifying selection and positive selection, respectively. When compared with S. sclerotiorum , fewer secreted effector proteins were found in C. shiraiana . The number of genes involved in pathogenicity, including secondary metabolites, carbohydrate active enzymes, and P450s, in the C. shiraiana genome was comparable with that of other necrotrophs but higher than that of biotrophs and saprotrophs. The growth-related genes and plant cell-wall-degradation-related genes in C. shiraiana were expressed in different developmental and infection stages, and may be potential targets for prevention and control of this pathogen. These results provide new insights into C. shiraiana pathogenic mechanisms, especially host range and necrotrophy features, and lay the foundation for further study of the underlying molecular mechanisms.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Serratiopeptidase (EC 3.4.24.40), a proteolytic enzyme, is one of the most promising enzymes being used in biopharmaceutical industry. Mulberry phyllosphere, being an unexplored niche for exploration of protease production, was chosen for the present study. Protease producing bacteria were isolated from the tissues of mulberry plant as well as its rhizospheric soil. Two protease producing bacteria belonging to Serratia genus were found to be potential serratiopeptidase producers. Among them, the endophyte, i.e., Serratia marcescens MES-4 presented 95 Units/mL activity, while the soil isolate i.e., Serratia marcescens MRS-11 presented 156 Units/mL activity.

A strain of thiamethoxam-degrading endophyte, named TMX13, was isolated from roots of mulberry ( Morus alba L.) and was identified as Enterobacter sp. Inoculating Chinese cabbage ( Brassica chinensis L) with strain TMX13- gfp ( gfp -labeled TMX13) could significantly reduce thiamethoxam residues in the aboveground part (edible portion) of the vegetable. The theoretical daily intake (TDI) of thiamethoxam via consumption of TMX13- gfp inoculated Chinese cabbage was 0.17 μg/kg body weight per day, far less than the prescribed acceptable daily intake (ADI) for this pesticide. TMX13- gfp colonization could increase the leaf chlorophyll content and plant biomass and promote the development of plant roots. Compared with the uninoculated treatment, the contents of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) and the activity of superoxide dismutase (SOD) in leaves of the TMX13- gfp inoculated plants decreased by 18.4%-60.2%, suggesting that TMX13- gfp colonization could alleviate the oxidative stress induced by thiamethoxam exposure. The total amounts of organic acids and amino acids in root exudates from the TMX13- gfp inoculated Chinese cabbage decreased by 9.2% and 85.2%, respectively. Results of this study lead to the conclusion that the isolated endophyte Enterobacter sp. TMX13 could reduce thiamethoxam residues in edible vegetables, promote plant growth, and alleviate the phytotoxic effects induced by thiamethoxam exposure.

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal)., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mulberry sclerotiniose caused by Ciboria shiraiana is a devastating disease of mulberry ( Morus alba L.) fruit in Northwest China. At present, no disease-resistant varieties are used in production, as the molecular mechanisms of this disease are not well understood. In this study, to explore new prevention methods and provide direction for molecular breeding, transcriptomic sequencing and un-targeted metabolomics were performed on healthy (CK), early-stage diseased (HB1), and middle-stage diseased (HB2) mulberry fruits. Functional annotation, gene ontology, a Kyoto encyclopedia of genes and genomes (KEGG) analysis, and a Mapman analysis of the differentially expressed genes revealed differential regulation of genes related to plant hormone signal transduction, transcription factors, and phenylpropanoid biosynthesis. A correspondence between the transcript pattern and metabolite profile was observed in the phenylpropanoid biosynthesis pathway. It should be noted that the log2 ratio of eugenol (isoeugenol) in HB1 and HB2 are 85 times and 23 times higher than CK, respectively. Our study shows that phenylpropanoid biosynthesis may play an essential role in response to sclerotiniose pathogen infection and eugenol(isoeugenol) enrichment in mulberry fruit, which may provide a novel method for mulberry sclerotiniose control., Competing Interests: The authors declare no conflict of interest.

Woody plants have considerable application potential in the phytoremediation schemes, owing to their long-lived large biomass and prosperous root systems in heavy metal(loid)-contaminated soil. Under greenhouse conditions, the physiological response characteristics and phytoremediation possibility of Morus alba L. and its associated improvement of the bacterial and arbuscular mycorrhizal fungal (AMF) diversities in heavy metal(loid) co-contaminated soils were investigated. The results showed that the cultivated M. alba L. plant exhibited significant tolerance against the heavy metal(loid)s in co-contaminated soil and that the microbial diversities were improved notably. The contents of malondialdehyde (MDA) in M. alba L. leaves decreased with cultivation from 90 to 270 days, while the superoxide dismutase, peroxidase and catalase activities were maintained at normal levels to eliminate the production of lipid peroxides. The chemical compositions (e.g. amino acids, carbohydrates and proteins) in the root of M. alba L. fluctuated slightly throughout the cultivation period. Meanwhile, Cd, Pb and Zn were majorly concentrated in the M. alba L. roots, and the maximum contents were 23.4, 7.40 and 615.5 mg/kg, respectively. According to the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis results, the influence of M. alba L. on the rhizosphere AMF community was greater than that on the bacteria community. Meanwhile, the bacterial and AMF Shannon diversity indexes in the contaminated soil were enhanced by 18.7-22.0% and 7.14-16.4%, respectively, with the presence of M. alba L. Furthermore, the correlations between the availability of As, Cd, Pb, and Zn and Shannon diversity indexes of the bacterial and AMF communities were significantly (p < 0.05) positive with the phytoremediation of M. alba L. Therefore, M. alba L. can be suggested as a potential plant candidate for ecological remediation and for simultaneously improving the activity and diversity of microorganisms in contaminated soils.

Due to wide application of laccase, many researchers have shown great interest in over production of white-rot fungi laccase by co-culture. In this study, a white-rot fungus Trametes hirsuta SSM-3, and a yeast Sporidiobolus pararoseus SSM-8 were isolated and identified from Mulberry fruit. The capacity of S. pararoseus to enhance laccase production was remarkable in T. hirsuta , yielding 31777 ± 742 U/L, about 9.9 times higher than the result from the monoculture. The stimulatory factor in the S. pararoseus cells might be temperature-sensitive. The laccase production was enhanced by oil-extract of S. pararoseus and β-carotene induction. The amylase activity was decreased rapidly when strain S. pararoseus SSM-8 was inoculated. The glucose deprivation was occurred both in the mono-culture and co-culture process, and S. pararoseus propagated slowly in co-culture all the time. Native-PAGE revealed an increase of laccase-1(lac-1) level and a laccase-3 (lac-3) in the co-culture. Therefore, it was concluded that competition for resources between the co-cultured microbes leaded to amylase decreasing and the enhanced production of laccase. This conclusion was helpful for the development of laccase fermentation industry because it provided an effective, simple and economic method to improve the yield of laccase.

Endophytic bacteria-based biocontrol is regarded as a potential plant disease management strategy. Present study analyzed the diversity of mulberry endophytic bacteria basing on a culture-dependent approach and further evaluated their antimicrobial and plant growth-promoting (PGP) activities. A total of 608 cultivable endophytic bacteria, belonging to 4 phyla and 36 genera, were isolated from four mulberry cultivars having different resistance to sclerotiniosis in three seasons. Taxonomic compositional analysis results showed that Proteobacteria, Firmicutes, and Actinobacteria were the three dominant bacterial phyla in all communities, with the representative genera Pantoea, Bacillus, Pseudomonas, Curtobacterium, and Sphingomonas. Diversity analysis results indicated that the diversity of winter community was higher than that of spring or autumn, and higher diversities were detected in the resistant cultivar communities compared with the susceptible cultivar. Antagonism assays results showed that 33 isolates exhibited strong and stable activity against three phytopathogens which are Sclerotinia sclerotiorum, Botrytis cinerea, and Colletotrichum gloeosporioide. Eight endophytic bacteria were selected out from 33 antagonists based on the evaluation of antagonistic and PGP activities. Furthermore, pot experiment results revealed that all the 8 tested endophytes stimulated the growth of mulberry seedlings at different levels, and Bacillus sp. CW16-5 exhibited the highest promotion capacity, which the shoot length and the root fresh weight were increased by 83.37% and 217.70%, respectively. Altogether, present study revealed that mulberry harbors a large amount of diverse cultivable endophytic bacteria and they also serve as novel sources of beneficial bacteria and bioactive metabolites., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Brown spot disease caused by Colletotrichum species was found on leaves of mulberry ( Morus alba L.) in Dujiangyan, Sichuan Province, China. Fungal isolates from leaf lesions were identified as six Colletotrichum species based on morphological characteristics and DNA analysis of the combined sequences ITS, GAPDH , ACT , CHS-1 , TUB2 , and GS . These included Colletotrichum fioriniae , C. fructicola , C. cliviae , C. karstii , C. kahawae subsp. ciggaro , and C. brevisporum . Results showed that the most important causal agent of mulberry anthracnose was C. fioriniae , causing typical brown necrotic spots or streaks, followed by C. brevisporum , C. karstii , and C. kahawae subsp. ciggaro , whereas the two other species ( C. fructicola and C. cliviae ) showed no pathogenicity to mulberry. This study is the first report of these species associated with mulberry in China.

It's long been understood that antimicrobial properties are one of the most important function in the field of biomedicine. In this thesis, we introduce a new technique to functionalize bacterial cellulose (BC) with antimicrobial properties by in situ method. We design a series of experiments on hydrolyzing mulberry leaves and exploring the methods of fermenting and purifying to obtain a BC complex with antimicrobial properties. Meanwhile, the anti-bacterial performances of MH-BC (fermented by the mulberry leaves acid hydrolysate fermentation medium) were evaluated with Escherichia coli and Staphylococcus aureus, and the experimental results showed that the MH-BC have excellent anti-bacterial activities. Considering the excellent biocompatibility of MH-BC towards hMSCs, we expect that this antibacterial functional BC composite will find potential applications in biomedicine and regenerative medicine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, the effects of intercropping with alfalfa and nitrogen application on the functional diversity of soil microbial community in mulberry rhizosphere were examined by Biolog-Ecoplate TM technique, and principal component and canonical analyses. Compared to monoculture with no nitrogen (N) addition, monoculture with N application and intercropping with alfalfa remarkably reduced soil pH and significantly increased the contents of soil organic matter, soil available N, soil water content, and activities of peroxidase and urease. Monoculture with N application and intercropping with alfalfa (with or without N application) increased the AWCD values, diversity index, and the carbon source utilization ratios of soil microbes. Higher increments of these parameters were detected in the treatment of intercropping plus N application. The results of principal component analysis showed that N application and intercropping changed the capacity of the rhizosphere microbial community for utilizing carbon sources. The utilization of carbon sources highly related to the principal components by the rhizosphere microbial communities was similar in the treatments of monoculture with N application and intercropping without N application. The utilization of itaconic acid and D-glucamaminic acid in the latter was more than 4% and was significantly higher than that in the former. The results from redundancy analysis showed that the soil microbial diversity in mulberry rhizosphere of the treatment of monoculture without N application was positively correlated with polyphenol oxidase activity and negatively correlated with soil water content, whereas that of monoculture with N application and intercropping without N application was significantly positively correlated with soil pH and soil water content and negatively correlated with soil N avalaibility. The diversity of the microbes in the rhizosphere soil of mulberries under the treatment of intercropping with N application showed positive correlation with soil N availability and was significantly negatively correlated with soil pH.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0 × 10 6 and 1.0 × 10 5  CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

The aim of this work was to gain insights into the molecular mechanisms and dynamics of the mulberry (Morus atropurpurea) fruit response to Ciboria carunculoides infection. A transcriptomic and proteomic study was carried out based on RNA sequencing and isobaric tags for relative and absolute quantification analysis, respectively. These data were then validated using quantitative real-time PCR and multiple reaction monitoring assays. Comparative analyses revealed that 9.0% of the transcriptome and 20.8% of the proteome were differentially regulated after C. carunculoides infection at the early stage (stage 1) and middle stage (stage 2), but correlation analysis revealed that only 145 genes were differentially regulated at both the transcriptome and proteome levels. The combined transcriptome and proteome analysis showed that plant hormone signal transduction, calcium-mediated defense signaling, transcription factors, and secondary metabolites were stimulated, whereas photosynthesis and cellular growth-related metabolism were suppressed after C. carunculoides infection. These finding provide theoretical foundation for disease resistance breeding of C. carunculoides. BIOLOGICAL SIGNIFICANCE: Ciboria carunculoides is a major fungal pathogen that infects mulberry fruit, leading to extensive damage and productivity loss. Despite this major impact, the mulberry fruit response to C. carunculoides infection has yet to be characterized. This study provides the first system-wide datasets with which to examine changes in the transcriptome and proteome after C. carunculoides infection in mulberry fruit. The results showed that plant hormone signal transduction, calcium-mediated defense signaling, and other pathways were stimulated, whereas photosynthesis and cellular growth-related metabolism were suppressed by C. carunculoides. These results will lead to a better understanding of the molecular mechanisms triggered in mulberry fruit in response to C. carunculoides infection and will provide new molecular targets for regulating defense responses to fungal pathogens in berry fruits., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 ( MuMLPL329 ) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea , Pseudomonas syringae pv tomato DC3000 ( Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants., (© 2018 Gai et al.)

Outbreaks of root rot disease in the productive South Indian sericulture belt have threatened the sustainability of the industry. Macrophomina phaseolina (Tassi) Goid. causing charcoal rot is the predominant pathogen to which all productive mulberry cultivars are susceptible. The present study was undertaken to identify molecular markers associated with charcoal rot resistance in mulberry. A mapping panel comprising 214 diverse entries from the Indian germplasm collection was assessed for charcoal rot resistance by artificial inoculation. Resistance to the pathogen was observed in 20 entries, and 51 were found to be moderately resistant. A total of 773 alleles generated across 105 SSR loci and 20,384 AFLP markers generated using 32 EcoRI-NN and MseI-CNN primer combinations were used in genetic analysis. The panel was weakly structured with two subpopulations. However, most entries were found to be admixtures. Survival of cuttings and number of roots per sapling were associated with root rot resistance. Association mapping was performed using different linear mixed models. Five AFLP markers explaining 9.6-12.7% of the total phenotypic variance were found to be significantly (p < 0.05) associated with root rot resistance. Significant associations were also detected in four AFLP markers for survival of cuttings, and these markers explained 10.7-14.2% of the total phenotypic variance. These markers should be validated using mapping populations derived from contrasting biparental combinations by linkage analysis for use in marker-assisted gene pyramiding for durable resistance. The resistant genotypes identified in this study will substantially contribute to genetic improvement of mulberry for charcoal rot resistance and can be integrated into conventional breeding programmes., Competing Interests: The authors have declared that no competing interests exist.

This study was conducted to investigate the effect of lactic acid bacteria (LAB) strains on color properties, phenolic profile and antioxidant activities of mulberry juice. Mulberry juice was separately fermented at 37 °C for 36 h using Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus paracasei. The results showed that lactic acid fermentation impacted on the color of the juice. Moreover, the study demonstrated that LABs impacted on the phenolic profile of the juice. Syringic acid, cyanidin-3-O-rutinoside and quercetin were the predominant phenolic acid, anthocyanin and flavonol respectively in the lactic-acid-fermented mulberry juice. The degree of radical scavenging activity was species-specific with the L. plantarum fermented juice having the highest radical scavenging activities. The correlation analysis demonstrated that flavonols and anthocyanins were mostly responsible for the increased in 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity while phenolic acids and flavonols were responsible for 2,2-diphenyl-1-picrylhydrazyl scavenging activity and reducing power capacity of the fermented juice., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A better understanding of tree-based intercropping effects on soil physicochemical properties and bacterial community has a potential contribution to improvement of agroforestry productivity and sustainability. In this study, we investigated the effects of mulberry/alfalfa intercropping on soil physicochemical properties and soil bacterial community by MiSeq sequencing of bacterial 16S rRNA gene. The results showed a significant increase in the contents of available nitrogen, available phosphate, available potassium, and total carbon in the rhizosphere soil of the intercropped alfalfa. Sequencing results showed that intercropping improved bacterial richness and diversity of mulberry and alfalfa based on richness estimates and diversity indices. The relative abundances of Proteobacteria, Actinobacteria, and Firmicutes were significantly higher in intercropping mulberry than in monoculture mulberry; and the abundances of Proteobacteria, Bacteroidetes, and Gemmatimonadetes in the intercropping alfalfa were markedly higher than that in monoculture alfalfa. Bacterial taxa with soil nutrients cycling were enriched in the intercropping system. There were higher relative abundances of Bacillus (0.32%), Pseudomonas (0.14%), and Microbacterium (0.07%) in intercropping mulberry soil, and Bradyrhizobium (1.0%), Sphingomonas (0.56%), Pseudomonas (0.18%), Microbacterium (0.15%), Rhizobium (0.09%), Neorhizobium (0.08%), Rhodococcus (0.06%), and Burkholderia (0.04%) in intercropping alfalfa soil. Variance partition analysis showed that planting pattern contributed 26.7% of the total variation of bacterial community, and soil environmental factors explained approximately 56.5% of the total variation. This result indicated that the soil environmental factors were more important than the planting pattern in shaping the bacterial community in the field soil. Overall, mulberry/alfalfa intercropping changed soil bacterial community, which was related to changes in soil total carbon, available phosphate, and available potassium., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

A wide range of miRNAs have been identified as phloem-mobile molecules that play important roles in coordinating plant development and physiology. Phytoplasmas are associated with hundreds of plant diseases, and the pathogenesis involved in the interactions between phytoplasmas and plants is still poorly understood. To analyse the molecular mechanisms of phytoplasma pathogenicity, the miRNAs profiles in mulberry phloem saps were examined in response to phytoplasma infection. A total of 86 conserved miRNAs and 19 novel miRNAs were identified, and 30 conserved miRNAs and 13 novel miRNAs were differentially expressed upon infection with phytoplasmas. The target genes of the differentially expressed miRNAs are involved in diverse signalling pathways showing the complex interactions between mulberry and phytoplasma. Interestingly, we found that mul-miR482a-5p was up-regulated in the infected phloem saps, and grafting experiments showed that it can be transported from scions to rootstock. Based on the results, the complexity and roles of the miRNAs in phloem sap and the potential molecular mechanisms of their changes were discussed. It is likely that the phytoplasma-responsive miRNAs in the phloem sap modulate multiple pathways and work cooperatively in response to phytoplasma infection, and their expression changes may be responsible for some symptoms in the infected plants.

Chalcone synthase (CHS) is an essential enzyme in the phenylpropanoid pathway that catalyzes the first step in flavonoid biosynthesis in plants under diverse environmental stress. We have used CHS as a candidate gene in mulberry and developed Single Nucleotide Polymorphism (SNP) based co-dominant Cleaved Amplified Polymorphic Sequence (CAPS) marker associated with the CHS locus. The segregation pattern of the marker was studied in an F1 population derived from a hybridization program between two mulberry genotypes showing polymorphism for the CHS locus. Differential CHS activity of the recombinants has been correlated with the segregation pattern of the marker. Homology modelling and docking studies are performed for both the identified CHS alleles and correlated with respective CHS activity. Phenotyping of Powdery Mildew infected F1 population indicated a probable association with the CAPS marker.

The objective of the present study was to evaluate the effect of mulberry leaf extract (ME) fermented with Lactobacillus acidophilus A4 (A4) on intestinal mucositis induced by 5-fluorouracil (5-FU) in a rat model. Male Wistar rats were gavaged with A4, ME, fermented mulberry leaf extract FME) or lafutidine (LAF) for 10 days and injected intraperitoneally with 5-FU (150 mg kg -1 ) or saline (normal control) on day 7 to induce mucositis. After euthanizing the animals, their small and large intestines were removed for evaluation of histopathologic parameters, myeloperoxidase (MPO) activity, mucin content, and mRNA expression of the mucin gene and pro-inflammatory cytokine interleukin (IL)-1β. 5-FU induced significant weight loss, shortened villi height, and increased histological severity, IL-1β expression, and MPO activity compared to the normal control group. These pathological changes were markedly ameliorated by treatment with A4, ME and FME. These treatments also stimulated MUC2 and MUC5AC gene expression and mucin production, and reduced IL-1β expression and MPO level. Interestingly, FME had the greatest protective effect on 5-FU-induced mucositis in rats., Significance and Impact of the Study: Our results suggest that fermented mulberry leaf extract (ME) may provide synergistic therapeutic benefits of both probiotics and natural plant extracts in prevention of 5-fluorouracil-induced mucositis. These impacts are particularly significant given the induction of MUC2 and MUC5AC gene expressions for production of mucins and the reduction of pro-inflammatory cytokine interleukin-1β in gut environments. Therefore, we proposed that enhanced functionality of ME by fermentation of Lactobacillus acidophilus A4 can be applied as food-grade adjuncts for mucositis therapy and prevention in food industry., (© 2017 The Society for Applied Microbiology.)

To alleviate adverse effects of heavy metal toxicity, diverse range of removing methods have been suggested, that is usage of algae, agricultural by-products and microorganisms. Here, we investigated lead (Pb) biosorption efficacy by two lactic acid bacteria species (LABs) in broiler chickens. In an in vitro study, Pb was added to culture medium of LABs (Lactobacillus pentosus ITA23 and Lactobacillus acidipiscis ITA44) in the form of lead acetate. Results showed that these LABs were able to absorb more than 90% of Pb from the culture medium. In follow-up in vivo study, LABs mixture was added to diet of broiler chickens contained lead acetate (200 mg/kg). Pb exposure significantly increased lipid peroxidation and decreased antioxidant activity in liver. The changes were recovered back to normal level upon LABs supplementation. Moreover, addition of LABs eliminated the liver tissue lesion and the suppressed performance in Pb-exposed chicks. Analysis of liver and serum samples indicated 48% and 28% reduction in Pb accumulation, respectively. In conclusion, results of this study showed that L. pentosus ITA23 and L. acidipiscis ITA44 effectively biosorb and expel dietary Pb from gastrointestinal tract of chickens.

Three Agrobacterium rhizogenes strains were tested for their ability to transform the plant Mois alba L. and to induce production of the secondary metabolites betulin and betulinic acid. All the tested strains of A. rhizogenes (R1601, LBA9402 and R1000) were able to induce hairy root formation in leaf tissue explants. Strain LBA9402 had the highest rate of infection (92.7% ± 8.8%), whereas strain R1601 had the lowest rate (87.4% ± 9.3%). The highest number of hairy roots per explant (5.6 ±0.5) and the greatest root length (2.4 ± 0.2 mm) were obtained with strain LBA9402. We also evaluated dry weight (a measure of growth) and betulin and betulinic acid production in hairy roots and found that the highest growth (167.8 ± 14.5 mg/flask) occurred after infection with strain LBA9402. Furthermore the highest production of betulin (5.4 ± 0.4 mg/g dry weight) and betulinic acid (2.3 ± 0.2 mg/g dry weight) was noted using strain LBA9402. Among three elicitors, yeast extract showed the highest induction of betulin production (8.7 ± 0.4 mg/g) and silver nitrate induced the highest yield of betulinic acid (4.1 ± 0.2 mg/g). Our study showed that A. rhizogenes strain LBA9402 was the most effective of the three tested strains for production-of transformed root cultures and betulin and betulinic acid.

Objective: We studied the biological and the epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, which is a devastating fungal disease of mulberry., Methods: We studied the asexual and sexual reproductive phase stages of C. shiraiana, including the infection ability of hyphal, dormancy of sclerotia, the structures, release, number and germination of ascospores from apothecia, as well as the phenology of sclerotial germination., Results: In C. shiraiana, hyphae had no infection ability toward the female flowers of mulberry. Sclerotia of C. shiraiana must undergo cold treatment above 6 weeks, then the dormancy-breaking sclerotia could germinate to apothecia. One to fifteen apothecia were germinated from one sclerotium, and the number of ascospores in a 1.5 cm diameter apothecia could contain up to (5.6-6.3)×107. Ascospore C. shiraiana had significantly higher germination rates in acid than in neutral and alkaline environments. From late January to middle April, sclerotia germinated to apothecia, and got the highest value in the middle of March., Conclusion: C. shiraiana is a formidable pathogen to cause epidemic disease and damage in mulberry.

Scleromitrula shiraiana causes the popcorn disease in mulberry trees resulting in severe economic losses. Previous studies have shown that melanin may play a vital role in establishing the pathogenicity of fungi. In the present study, we identified the melanin produced in S. shiraiana belongs to DHN melanin by gas chromatography-mass spectrometry, and cloned the laccase Sh-lac, a potential DHN melanin biosynthesis gene from S. shiraiana. We obtained two stable Sh-lac silenced transformants using RNAi, ilac-4 and 8 to elucidate the DHN melanin biosynthetic pathway in S. shiraiana. The melanin production of ilac-4 and ilac-8 was significantly reduced, and their vegetative growth was also suppressed. Results such as these led to a proposal that Sh-lac played a key role in DHN melanin formation in S. shiraiana and may function differentially with other melanin biosynthetic genes. The inhibition of melanin was accompanied by the decrease of oxalic acid and the adhesion of hyphae was impaired. Our results indicated that laccase was an important enzyme in the synthesis of melanin and might play a critical role in the pathogenicity of S. shiraiana.

The aim of this study was to investigate the effects of high hydrostatic pressure and thermal processing on microbiological quality, bioactive compounds, antioxidant activity, and volatile profile of mulberry juice. High hydrostatic pressure processing at 500 MPa for 10 min reduced the total viable count from 4.38 log cfu/ml to nondetectable level and completely inactivated yeasts and molds in raw mulberry juice, ensuring the microbiological safety as thermal processing at 85 ℃ for 15 min. High hydrostatic pressure processing maintained significantly (p < 0.05) higher contents of total phenolic, total flavonoid and resveratrol, and antioxidant activity of mulberry juice than thermal processing. The main volatile compounds of mulberry juice were aldehydes, alcohols, and ketones. High hydrostatic pressure processing enhanced the volatile compound concentrations of mulberry juice while thermal processing reduced them in comparison with the control. These results suggested that high hydrostatic pressure processing could be an alternative to conventional thermal processing for production of high-quality mulberry juice.

Mulberry fruit sclerotiniosis is a common soil-borne disease. We hypothesize that there is a relationship between the incidence of mulberry fruit sclerotiniosis and the soil fungal community. Therefore four mulberry genotypes with different resistance to sclerotiniosis were selected to study their soil fungal community under the same condition. A total of 6 phyla and 93 genera were identified from all mulberry samples. Genera affected by genotype, cover, and genotype by cover interactions, were 23, 20, and 11, respectively. There were 10 genera that differed between the resistant cultivars and the susceptible cultivars. In addition, the relative abundances of Humicola and Mortierella in the resistant mulberry cultivars with the cover treatments were significantly higher than that of in the susceptible cultivars. However, the relative abundance of Sclerotiniaceae and Scleromitrula that may cause sclerotiniosis in the uncover treatments were significantly higher compared with the cover treatments. The results suggested that the genotype of mulberry has a significant impact on the soil microbial community that may result in differences in resistance to sclerotiniosis, and covering could reduce the relative abundance of Sclerotiniaceae or Scleromitrula.

This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.

Previously, it was reported that Enterococcus mundtii (E. mundtii) was associated with flacherie disease of silkworm larvae reared on artificial diet. In this study, we report that E. mundtii was isolated from diseased silkworm larvae, and validated as a pathogenic bacterium of the animal. When silkworm larva was infected with 1.04 × 10⁶ colony-forming units of E. mundtii via oral administration of diet, half population died within six days, indicating that the bacterium is pathogenic to silkworm. Less severe infection was found to cause anorexia and hamper the development of larvae. This pathogen was found to proliferate in both time- and dose-dependent manner in the gastrointestinal tract of the animal. The bacterium was isolated from powder of artificial diet made from mulberry leaves, and from mulberry leaves growing at a field. Minimum inhibitory concentration determination revealed that this bacterium was susceptible to tested antibiotics. Vancomycin treatment of diet significantly decreased the number of E. mundtii in intestine of silkworm larvae infected with the bacteria, compared to control. Furthermore, autoclaving or gamma ray irradiation of diet was also effective for exclusion of E. mundtii from the diet without the loss of its nutrient capacities. These results suggest that mulberry leaves used in making artificial diet for silkworm larvae is one of the sources of E. mundtii infection; and that antibiotic treatment, autoclaving or gamma ray irradiation of artificial diet can exclude the bacteria.

The yellow dwarf disease associated with phytoplasmas is one of the most devastating diseases of mulberry and the pathogenesis involved in the disease is poorly understood. To analyze the molecular mechanisms mediating gene expression in mulberry-phytoplasma interaction, the comprehensive sRNA changes of mulberry leaf in response to phytoplasma-infection were examined. A total of 164 conserved miRNAs and 23 novel miRNAs were identified, and 62 conserved miRNAs and 13 novel miRNAs were found to be involved in the response to phytoplasma-infection. Meanwhile, target genes of the responsive miRNAs were identified by sequencing of the degradome library. In addition, the endogenous siRNAs were sequenced, and their expression profiles were characterized. Interestingly, we found that phytoplasma infection induced the accumulation of mul-miR393-5p which was resulted from the increased transcription of MulMIR393A, and mul-miR393-5p most likely initiate the biogenesis of siRNAs from TIR1 transcript. Based on the results, we can conclude that phytoplasma-responsive sRNAs modulate multiple hormone pathways and play crucial roles in the regulation of development and metabolism. These responsive sRNAs may work cooperatively in the response to phytoplasma-infection and be responsible for some symptoms in the infected plants.

To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography-mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H2O2 and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different., (© 2013 John Wiley & Sons Ltd.)

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.

Objective: Antagonistic endophytic strains with strongly inhibitory activity to mulberry bacterial blight (P. syringae pv. mori) were isolated from mulberry endophytes, we identified the antagonistic endophyte and optimized the fermentation conditions., Method: Streak plate method was used to separate the endophytes from healthy mulberry tissues after strict surface disinfection. Antagonistic endophytes were screened out through inhibition zone method. Strain SWg2 was identified by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment., Result: In total 77 endophytic strains have been isolated from healthy mulberry. SWg2 showed strong and stable antagonistic activity to mulberry bacterial blight. The morphological, cultural, physiological, and biochemical characteristics assays indicated that SWg2 belongs to Pantoea sp. The 16S rDNA sequence phylogenetic analysis reveals that SWg2 appeared a sister lineage to P. agglomerans. The optimized culture conditions of strain SWg2 were liquid volume 20 mL in 100 mL flask, 170 r/min at 28 degrees C, inoculation size of 4% for 5 d with a medium of 2.0% glycerol, 2.0% NH4NO3, 0.1% KH2PO4, 0.15% MgSO4 x 7H2O at initial pH of 7.5., Conclusion: The antagonistic endophytic strain SWg2 to mulberry bacterial blight was identified as P. agglomerans. SWg2 strain shows stronger antagonistic action to mulberry bacterial blight under optimized fermentation conditions.

A new isolate from apple (Malus domestica) producing dextransucrase and dextran was characterized by morphological, biochemical and 16S rDNA analyses. The bacterium that was identified as Weissella cibaria (GenBank accession number KC110687) was Gram positive, non-motile, short rod, catalase negative, vancomycin resistant. The maximum dextransucrase activity (5.8 U/ml) as well as dextran concentration of 38 mg/ml was observed at 24°C after 12 h of growth under static condition. The polyethylene glycol (PEG)-400 fractionation of partially purified enzyme showed a specific activity of 20.0 U/mg and displayed a single homogenous band of molecular size 177 kDa on sodium dodecyl sulphate-Poly acrylamide gel electrophoresis (SDS-PAGE) (7.5%). The dextran was enzymatically synthesized using dextransucrase and substrate sucrose. Dextran was purified by alcohol precipitation for further characterization. The rheological analysis of dextran elucidated a non-Newtonian pseudoplastic behaviour. (1)H and (13)C NMR analyses revealed the fact that the dextran contained d-glucose monomer with 93.0% of α(1 → 6) linear and 7.0% of α(1 → 3) branched linkages.

Proteome analysis is becoming a powerful tool in the functional characterization of organisms, and takes a broad, comprehensive, systematic approach to understanding biology. Following the sequencing of the phytoplasma genomes, the next step is to characterize the expressed proteome of phytoplasmas to acquire the verification and functional annotation of all predicted genes and their protein products. Here, we describe the protocol of mulberry dwarf phytoplasma purification, phytoplasma protein extraction and separation by SDS-PAGE, in-gel tryptic digestion of the proteins, separation of the digested peptides by liquid chromatography, and identification of the peptides by mass spectrometry. The protocol described here is also applicable to the analysis of other phytoplasma proteomes.