Identification of Resistance to Pathogenesis Related Protein GmPR1L in Tobacco Botrytis cinerea Infection

Soybean(Glycine max(Linn.)Merr.)annual leguminous crop is cultivated all over the world.The occurrence of diseases has a great impact on the yield and quality of soybean.In this study,based on the RNA-seq of soybean variety M18,a complete CDS(Coding sequence)GmPR1L of the pathogenesis-related protein 1 family was obtained,which has the ability to resist fungal diseases.The overexpression vector and interference expression vector were transferred into tobacco NC89,and the resistance of transgenic tobacco(Nicotiana tabacum L.)to Botrytis cinerea infection was identified.The results show that:Compared with the control,the activities of related defense enzymes SOD(Superoxide dismutase),POD(Peroxidase),PAL(L-phenylalanine ammonia-lyase)and PPO(Polyphenol oxidase)in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees,and increased significantly at different infection time points.The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains.The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line,and the disease spot rate of OEA2 was significantly lower than that of the control line.The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line.It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B.cinerea.

The Herbicidal Activity of Mutant Isolates from Botrytis cinerea

Fifteen mutant isolates were obtained by ultraviolet mutation from parent isolate Botrytis cinerea BC-4. Among them three mutant isolates, BC4-1, BC4-2, and BC4-15, showed strong herbicidal activity. BC4-1 showed maximum herbicidal activity for inhibition of germination and growth of Digitaria sanguinalis L. and Arnaranthus retroflexus L. The results also showed that herbicidal activity was influenced by differing pH of PD media, with pH value of 4.0 being the optimum. The crude toxin was extracted using chloroform, petroleum ether, and ethyl acetate, respectively, and the ethyl acetate extracts showed the strongest inhibitory activity on the germination and growth of D. sanguinalis L. and A. retroflexus L. Using HPLC, one fraction with an absorption peak at 271 nm was separated from the crude toxin. This fraction could strongly inhibit the growth of D. sanguinalis L. at a concentration of 100 mg L^-1 and could completely inhibit the seed germination of D. sanguinalis L. and A. retroflexus L. at a concentration of 50 mg L^-1 .

Hot air treatment activates defense responses and induces resistance against Botrytis cinerea in strawberry fruit

The effect of hot air（HA, 45°C, 3.5 h） treatment on reducing gray mold caused by Botrytis cinerea in strawberry fruit and the possible mechanisms were investigated. The results showed that HA treatment significantly reduced lesion diameter and enhanced activities of chitinase（CHI）, β-1,3-glucanase and phenylalanine ammonia-lyase（PAL） in strawberry fruit. Total phenolic contents were also increased by HA treatment. The activities of antioxidant enzymes including superoxide dismutase（SOD）, catalase（CAT） and ascorbate peroxidase（APX） were higher in HA treated strawberry fruit than those in control. Expression of three defense related genes such as CAT, CCR-1 allele and PLA6 was greatly induced in HA treated strawberry fruit with or without inoculation by B. cinerea. In addition, the in vitro experiment showed that HA treatment inhibited spore germination and tube growth of B. cinerea. These results suggested that HA treatment directly activated disease resistance against B. cinerea in strawberry fruit without priming response and directly inhibiting growth of B. cinerea.

Effects of different LED light wavelengths on the resistance of tomato against Botrytis cinerea and the corresponding physiological mechanisms

New types of light-emitting diode （LED） sources were applied to irradiate Botrytis cinerea mycelium and tomato leaves that were inoculated with B. cinerea to assess the effect of different LED light wavelengths on the infection of tomato with B. cinerea, to determine the optimum light wavelengths to control B. cinerea, and to explore the mechanism of LED influ- ence on the development of gray mold. The results showed that purple light and blue light irradiation significantly inhibited the growth of B. cinerea mycelium, and the inhibition rates were 22.3 and 15.16%, respectively, and purple light exhibited a better inhibitory effect than blue light. The lesion development of B. cinerea on tomato leaves was significantly inhibited upon irradiation with red and purple light with inhibition rates of 32.08 and 36.74%, respectively. Irradiation with red light inhibited the oxidative burst of superoxide anion （O2-） that was caused by infection with B. cinerea, and red light regulated the H2O2 content in the tomato leaf, which increased and rapidly returned to a lower level. In addition, red light irradiation improved the activity of superoxide dismutase （SOD）, catalase （CAT） and peroxidase （POD） in tomato leaves. However, purple light irradiation did not make tomato leaves exhibit this kind of physiological response. Therefore, red light and purple light can suppress gray mold in tomatoes, but the disease suppression mechanisms of these two types of LED light are different. In general, red light suppresses gray mold primarily by regulating the tomato defense mechanism for disease, whereas the suppression of tomato gray mold by purple light can be partially explained by the photo-inhibition of B. cinerea.

Plastic bronchitis associated with Botrytis cinerea infection in a child:A case report

BACKGROUND Plastic bronchitis(PB)frequently occurs in children after the surgical repair of congenital cardiac defects or in the presence of inflammatory or allergic diseases of the lung.Accurate epidemiological data of this condition are still lacking.CASE SUMMARY A 5-year-old boy,with a clear medical history,presented to our hospital with persistent cough and pneumonia with segmental atelectasis on chest computerized tomography.He showed no significant improvement after 1 wk of amoxicillin-clavulanate potassium treatment.Bronchial casts were extracted using flexible bronchoscopy.Pathological examination of the dendritic cast confirmed the diagnosis of type I PB.Botrytis cinerea was detected by next-generation sequencing of the bronchoalveolar lavage fluid.After the removal of the airway obstruction and fluconazole treatment,the patient recovered and was discharged 14 d after admission without the recurrence of cough.CONCLUSION Botrytis cinerea pneumonia should be considered in children with PB who still have prolonged cough and atelectasis after a regular course of antibiotic therapy.Flexible bronchoscopy and etiological examination should be performed in a timely manner to determine the diagnosis,clear the airway obstruction,and target etiological treatment.

An ABAGS-Like Metabolite of Botrytis cinerea Isolate BC4 and Its Inhibitory Activity to Seed Germination of Weeds

Aimed at finding out natural compounds to kill weeds, a plant pathogenic fungus, Botrytis cinerea isolate BC4, was chosen as a source. A significantly polar metabolite, which was different in chromatographic behavior from ABAGE-like metabolite, was isolated from cultural filtrate of the fungus by column chromatography on silica gel, preparative LC, TLC and HPLC. The structure of the metabolite was determined by HPLC-ESI MS, g.l.c, IR, ^1H NMR and hydrolysis. The results showed that this metabolite was quite similar with ABAGS. The bioassay showed the metabolite had inhibition to seed germination of both broadleaf weeds [Amaranthus retroflerus L. and Chenopodium album L.] and gramineae weeds [Digitaria sanguinalis L. Scop and Echinochloa crusgalii L. Beauv]. It also had inhibitory activity to the seedling growth of broadleaf weeds. The bioassay, using Amaranthus retroflexus L. as an indicator, showed that the lowest concentration of the metabolite to inhibit root＋sprout growth of A. retroflexus was 0.6 μM, and the concentration for 50% inhibition was 1.3μM.

An ABAGE-Like Metabolite of Botrytis cinerea Isolate BC4 Inhibited the Growth of Hypocotyls and Roots of Amaranthus retroflexus Seedlings

A metabolite, which had an inhibitory effect on plant growth, was isolated from cultural filtrate of Botrytis cinerea isolate BC4 by column chromatography on silica gel and preparative HPLC. Its structure was determined from HPLC-ESI MS, GC, IR, ^1H NMR and ^13C NMR spectral data, as well as chemical hydrolysis. The results showed that this metabolite was quite similar to abscisic acid-β-D-glucopyranosyl ester（ABAGE） in structure. The inhibitory effect of the ABAGE-like metabolite on plant growth was investigated using a weed Amaranthus retroflexus L. as a bioassay material. The results showed that it inhibited hypocotyls and roots growth of A. retroflexus （Amaranthus retroflexus L.） seedlings when its concentration was over 0.5 μM. The concentrations for 50% inhibition of hypocotyls and roots growth of A. retroflexus seedlings were 2.8 and 1.4 μM, respectively.

Control Effect of Bacillus cereus CGMCC4348 against Botrytis cinerea and Its Mechanism

[ Objectivel The paper was to study antifungal activity and control mechanism of Bacillus cereus CGMCCA348 against Botrytis cinerea, and further find new methods against the disease. [Method] By quantitative bioassay, in vitro experiment and pot test, the antagonistic protein was extracted with ammonium sul- fate precipitation fractionation to determine the control efficiency. ECso of B. cereus CGMCC4348 was 6. 19 mg/L. Average controlling effect of B. cereus CGM- CC4348 on B. cinerea was 75.8%, equal to the control group which was treated by 50 mg/L pyrimethanil. Bacillus cereus CGMCCA348 had good inhibitory effect against B. cinerea. [ Conclusion] The paper provides scientific basis for green control technology against solanaceous vegetable diseases.

In field control of Botrytis cinerea by synergistic action of a fungicide and organic sanitizer

A new Integrated Pest Management program based on the combination of synthetic pesticide with a GRAS （generally recognized as safe）-classified sanitizer for the control of Botrytis cinerea in field conditions was described. The aim behind this research was to determine whether the use of this mixture would enhance the efficiency of pesticides while decreasing the recommended dose. Naturally infected tomato plants, grown in the greenhouse, were treated with two commonly used fungicides SWITCH （Syngenta, Switzerland） and SIGNUM （BASF, Germany） each alone or combined with a commercially available organic sanitizer PERACLEAN？5 （Evonik Industries, Germany）. A total of 27 treatments were tested consisting of three concentrations of synthetic fungicide （×1, ×1/2 and ×1/4 of the recommended dose） either applied separately or combined with three concentrations of the tested sanitizer （0.5, 1 and 1.5％）. The control efficacy achieved by the fungicides applied alone ranged between 0 and 66.7％ while all fungicide-sanitizer mixtures resulted in up to 70％ control of grey mould. The treatment that provides the maximum control of B. cinerea was the result mixture of ×1/4 of the recommended concentration of SWITCH （15 g L-1） with 0.5％ of PERACLEAN？5. This combination suppressed 85％ of grey mold infections while decreasing the usually used amount of this pesticide by 75％, reducing therefore the well known negative impacts of chemical pesticides on environment and consumers health.

Identification of Damage on Different Plants Caused by Botrytis cinerea and its Extracellular Macromolecular Toxin

With Bowytis cinerea and its extracellular macromolecular toxins as the test materials, 30 speiees of plants belonging to 29 genera and 21 families were selected as the test plants to observe the infectivity of B. dnerea and damage status of macromolecular toxins secreted by B. cinerea on plants. The resulsts showed that 17 species of plants were beth infected by B. cinerea and damaged by toxins, accounting for 56.7% of the total plants. Two species of plants could be neither infected by B. cinerea nor damaged by toxins. The study provided the reference for further understanding of pathogenic mechanism of plant pathogenic fungi toxins.

Tomato SlPti5 plays a regulative role in the plant immune response against Botrytis cinerea through modulation of ROS system and hormone pathways

While SlPti5 has been shown to play a crucial role in the regulation of antagonistic genes in Solanum lycopersicum and Arabidopsis against pathogen infection,there have been no comprehensive studies on the effects of SlPti5 on the regulatory response mechanism of reactive oxygen species(ROS) system and hormone pathways during growth and disease resistance of tomato plants.Here,we investigated the function of SlPti5 in the defense response of tomato against Botrytis cinerea utilizing a virus-induced gene silencing(VIGS)-based system.Expression profile analysis showed that SlPti5 was significantly induced upon B.cinerea infection,with high expression levels in the leaves and fruit of tomato.VIGS-based silencing of SlPti5 inhibited early vegetative growth,increased the plant’s susceptibility to infection,promoted the development of ROS,affected the expression of genes involved in the ROS scavenging system,and attenuated the expression of genes associated with pathogenesis and the ethylene/jasmonic acid signaling pathways.In sum,our data demonstrated that SlPti5 stimulates the immune response of tomato plant to Botrytis cinerea infection by involving the ethylene(ET)-and jasmonic acid(JA)-mediated pathways and modulating the expression of some key pathogenesis-related(PR) genes.

Physiological Characteristics of Boscalid Against Botrytis cinerea from Strawberry

A total of 96 monoconidial Botrytis cinerea strains were isolated from strawberry field.Physiological characteristics and resistance analysis to boscalid were investigated.Most isolates showed resistant phenotype and the resistance frequency of highly resistant isolates was as high as 45.10%.The effective concentration that inhibited conidia germination by 50% relative to the control values for sensitive isolates ranged from 0.068 to 60.56μg·mL^-1 in conidia germination assays.Compared to the sensitive strains,the boscalid-resistant strains had a significant increase in phenylalanine ammonia lyase(PAL)and peroxidase(POD)activity and more sensitivity to boscalid pressure.Fitness analysis showed statistically significant difference between sensitive and resistance isolates.Sequencing of SdhB gene indicated that a single mutation corresponded to His272.The results suggested that resistance to boscalid in B.cinerea populations could result in failure to control gray mold.These results would enrich the understanding of the resistance mechanism of B.cinerea to boscalid.

Host-targeting-motif Harbored Secretary Proteins in Genome of Plant Pathogenic Fungus Botrytis cinerea

According to our previous study, saprophytic fungi Botrytis cinerea contained 579 predicted secretary proteins. Among them, we found that 122 of these proteins contained the highly conserved pathogenic-related host-targeting-motif RxLx within 100 residues adjacent to the signal peptide cleavage site. According to PEDNAT and COG of the GenBank database, the functions of this motif containing proteins included metabolism modification and cell secretion. We blasted them in GenBank and found 47.54% had highly conserved homologues in other species, among them 74.1% had putative functional domains. This suggests these proteins are presumably ancient and vertically transmitted within the species. Many of these domains belonged to proteins which played roles in the pathogenic process of other kinds of pathogens and some had already been proved to be pathogenic secretary proteins of Botrytis cinerea. So we postulated that proteins contained host-targeting-motif RxLx were candidates participating in the pathogenesis of Botrytis cinerea.

Isolation and Identification of Botrytis cinerea in Processing Tomato(Solanum lycopersicum)

[Objective]The paper was to isolate and identify Botrytis cinerea from processing tomato.[Method]The strain nky007a was isolated and purified from fruits infected by tomato grey mold.After identification,it was determined as B.cinerea.The 18S rDNA and internal transcriptional spacer(ITS)sequences of strain nky007a were amplified for molecular identification and classification.[Result]The 18S rDNA sequence of the strain shared the homology of 99.95%with Botryotinia fuckeliana(EF 110887.1);the ITS rDNA sequence had the homology of 100%with Botryotinia fuckeliana(AB444949.1),and there was no difference in bases.Phylogenetic tree analysis of strain nky007a indicated that nky007a belonged to Botrytis.When healthy tomatoes were infected by the strain,the incidence rate of tomato grey mold was up to 95%,indicating the pathogenicity of the strain.[Conclusion]Morphological identification,molecular identification and pathogenicity analysis results show that the strain nky007a is a highly pathogenetic strain B.cinerea.

Safe antifungal lipopeptides derived from Bacillus marinus B-9987 against grey mold caused by Botrytis cinerea

Agricultural application studies, including field experiments and acute toxicity tests, were conducted for lipopeptides secreted by marine-derived Bacillus marinus B-9987. Benefiting from commercially available scaled-up lipopeptide purification, the sample of impurities（isolated from target lipopeptides）, raw extracted sample（purity： 9.08%）, partially purified sample（purity： 20.86%）, and highly purified sample（purity： 87.51%） were prepared from B. marinus B-9987 fermentation broth, and used in lab-scale antagonism tests, field experiments, swarming motility tests, and acute toxicity tests. Operations and conditions in field experiments were consistent with the Pesticide-Guidelines for the Field Efficacy Trials（GB/T 17980.28-2000）, and acute toxicity tests were executed according to Toxicological Test Methods of Pesticides for Registration（GB 15670-1995）. In agar diffusion tests in vitro and pot tests in vivo, all lipopeptide samples with different purities significantly inhibited Botrytis cinerea; meanwhile the sample of impurities isolated from target lipopeptides were not effective against B. cinerea. Results of lab-scale tests showed that the target lipopeptides were effective substances against B. cinerea. Thus, partially purified and raw extracted samples were used in field experiments instead of the highly purified sample for cost saving. In the field experiments against rose grey mold, biological control efficacy of 500 mg L–1 lipopeptides reached 67.53%, slightly lower than 74.05% reached by the agrochemical pyrimethanil. However, pyrimethanil severely suppressed B. marinus B-9987, whereas the lipopeptides promoted swarming motility and biocontrol efficacy of Bacillus biomass. Lipopeptides at 87.51% purity were tested for systemic acute toxicity and confirmed as low-toxicity substances. In conclusion, low-toxicity lipopeptides were potential alternatives to agrochemicals, and they also performed good promotion when combined with homologous biological control microorganism. There were 2 breakthroughs in this research：（1） marine-derived bacterial lipopeptides inhibited grey mold caused by B. cinerea in field experiments; and（2） purified bacterial lipopeptides（sample purity： 〉87.51%） were determined to be low-toxicity substances by systemic acute toxicity tests, satisfying the strict requirement of pesticide registration in China（required purity： 〉85%）. This study provides support for using extracellularBacillus-derived lipopeptides commercially similar to Bacillus-based biological control agents.

Enhanced resistance to Botrytis cinerea and Rhizoctonia solani in transgenic broccoli with a Trichoderma viride endochitinase gene

A endochitinase gene（Tch） from the fungus Trichoderma viride was introduced into broccoli（Brassica oleracea var. italica） by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.

Phytochrome B regulates jasmonic acid-mediated defense response against Botrytis cinerea in Arabidopsis

The phytochrome B mediated light signaling integrates with various phytohormone signalings to control plant immune response.However,it is still unclear whether phyB-mediated light signaling has an effect on the biosynthesis of jasmonate during plant defense response against Botrytis cinerea.In this study,we demonstrated that phyB-mediated light signaling has a role in this process.Initially,we confirmed that phyb plants were obviously less resistant to B.cinerea while phyB overexpressing plants showed significantly enhanced resistance.We also found that the expression of numerous JA biosynthesis genes was promoted upon treatment with red or white light when compared to that of darkness,and that this promotion is dependent on phyB.Consistent with the gene expression results,phyb plants accumulated reduced pool of JA-lle,indicating that phyB-mediated light signaling indeed increased JA biosynthesis.Further genetic analysis showed that light-mediated JAZ9 degradation and phyB-enhanced resistance were dependent on the receptor COI1,and that pifl/3/4/5(pi/q)can largely rescue the severe symptom of phyb.Taken together,our study demonstrates that phyB may participate in plant defense against B.cinerea through the modulation of the biosynthesis of JA.

BcSDR1 is involved in regulation of glucose transport and cAMP and MAPK signaling pathways in Botrytis cinerea

Botrytis cinerea is a typical necrotrophic pathogenic fungus that causes severe diseases in a wide range of plant species, leading to significant economic losses. Our previous study showed that BcSDR1 positively regulates growth,development, and pathogenicity of B. cinerea. However, the regulation mechanism of BcSDR1 and the relationship between BcSDR1 and cAMP and MAPK signaling pathways are not well understood. In this study, transcriptome data showed that BcSDR1 is involved in glucose transmembrane transport, signal transduction, secondary metabolism, and other biological processes. BcSDR1 mutant(BCt41) showed remarkably weak sensitivity to cAMP and MAPK signaling pathways specific inhibitors, SQ22536 and U0126, and significantly decreased cAMP content. The key genes of cAMP and MAPK signaling pathways, BcGB1, BcBTP1, BcBOS1, BcRAS1, and BcBMP3 were significantly upregulated,whereas BcPLC1, BcBCG1, BcCDC4, BcSAK1, BcATF1, and BcBAP1 were significantly downregulated(P<0.05).BcSDR1 was obviously upregulated in BcBCG2, BcBCG3, BcPKA1, and BcPKAR RNA interference(RNAi) mutants, but significantly downregulated in BcPKA2, BcBMP1, and BcBMP3 RNAi mutants. Thus, BcBCG2, BcBCG3, BcPKA1, and BcPKAR negatively regulate BcSDR1 expression, whereas BcPKA2, BcBMP1, and BcBMP3 positively regulate BcSDR1expression.

Identification and Analysis of miRNAs and siRNAs in Botrytis cinerea

Small RNAs in Botrytis cinerea were analyzed via high-throughput sequencing on BGISEQ-500 platform. A total of 8 novel miRNAs and 110 novel siRNAs were predicted. Sequence information, construction, length distribution, base bias and expression levels of miRNAs and siRNAs were determined as well. Through GO and KEGG enrichment analysis, the miRNA target genes are mostly located in membrane and organelle, possessed binding and catalytic activities, and involved in signal transduction and carbohydrate metabolism. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of B. cinerea at the transcriptional level.

Suppression of the homeobox gene HDTF_1 enhances resistance to Verticillium dahliae and Botrytis cinerea in cotton

Development of pathogen-resistant crops, such as fungus-resistant cotton, has significantly reduced chemical application and improved crop yield and quality. However, the mechanism of resistance to cotton pathogens such as Verticillium dahliae is still poorly understood. In this study, we characterized a cotton gene （HDTF1） that was isolated following transcriptome profiling during the resistance response of cotton to V. dahliae. HDTFI putatively encodes a homeodomain transcription factor, and its expression was found to be down-regulated in cotton upon inoculation with V. dahliae and Botrytis cinerea. To characterise the involvement of HDTF1 in the response to these pathogens, we used virusinduced gene silencing （VlGS） to generate HDTFl-silenced cotton. VIGS reduction in HDTF1 expression significantly enhanced cotton plant resistance to both pathogens. HDTF1 silencing resulted in activation of jasmonic acid （JA）-mediated signaling and JA accumulation. However, the silenced plants were not altered in the accumulation of salicylic acid （SA） or the expression of marker genes associated with SA signaling. These results suggest that HDTF1 is a negative regulator of the JA pathway, and resistance to V. dahliae and B. cinerea can be engineered by activation of JA signaling.