Transcriptomic Response of Clonostachys rosea Mycoparasitizing Rhizoctonia solani.

Clonostachys rosea is an important mycoparasitism biocontrol agent that exhibits excellent control efficacy against numerous fungal plant pathogens. Transcriptomic sequencing may be used to preliminarily screen mycoparasitism-related genes of C. rosea against fungal pathogens. The present study sequenced and analyzed the transcriptome of C. rosea mycoparasitizing a Basidiomycota (phylum) fungal pathogen, Rhizoctonia solani, under three touch stages: the pre-touch stage, touch stage and after-touch stage. The results showed that a number of genes were differentially expressed during C. rosea mycoparasitization of R. solani. At the pre-touch stage, 154 and 315 genes were up- and down-regulated, respectively. At the touch stage, the numbers of up- and down-regulated differentially expressed genes (DEGs) were 163 and 188, respectively. The after-touch stage obtained the highest number of DEGs, with 412 and 326 DEGs being up- and down-regulated, respectively. Among these DEGs, ABC transporter-, glucanase- and chitinase-encoding genes were selected as potential mycoparasitic genes according to a phylogenetic analysis. A comparative transcriptomic analysis between C. rosea mycoparasitizing R. solani and Sclerotinia sclerotiorum showed that several DEGs, including the tartrate transporter, SDR family oxidoreductase, metallophosphoesterase, gluconate 5-dehydrogenase and pyruvate carboxylase, were uniquely expressed in C. rosea mycoparasitizing R. solani. These results significantly expand our knowledge of mycoparasitism-related genes in C. rosea and elucidate the mycoparasitism mechanism of C. rosea.

The Adenylate Cyclase-Encoding Gene crac Is Involved in Clonostachys rosea Mycoparasitism.

Clonostachys rosea is an excellent biocontrol fungus against numerous fungal plant pathogens. The cAMP signaling pathway is a crucial signal transduction pathway in fungi. To date, the role of the cAMP signaling pathway in C. rosea mycoparasitism remains unknown. An adenylate cyclase-encoding gene, crac (an important component of the cAMP signaling pathway), was previously screened from C. rosea 67-1, and its expression level was dramatically upregulated during the C. rosea mycoparasitization of the sclerotia of Sclerotinia sclerotiorum. In this study, the function of crac in C. rosea mycoparasitism was explored through gene knockout and complementation. The obtained results show that the deletion of crac influenced the growth rate and colony morphology of C. rosea, as well as the tolerance to NaCl and H2O2 stress. The mycoparasitic effects on the sclerotia of S. sclerotiorum and the biocontrol capacity on soybean Sclerotinia stem rot in ∆crac-6 and ∆crac-13 were both attenuated compared with that of the wild-type strain and complementation transformants. To understand the regulatory mechanism of crac during C. rosea mycoparasitism, transcriptomic analysis was conducted between the wild-type strain and knockout mutant. A number of biocontrol-related genes, including genes encoding cell wall-degrading enzymes and transporters, were significantly differentially expressed during C. rosea mycoparasitism, suggesting that crac may be involved in C. rosea mycoparasitism by regulating the expression of these DEGs. These findings provide insight for further exploring the molecular mechanism of C. rosea mycoparasitism.

The Mitogen-Activated Protein Kinase Gene Crmapk Is Involved in Clonostachys chloroleuca Mycoparasitism.

Clonostachys chloroleuca is a mycoparasite used for biocontrol of numerous fungal plant pathogens. Sequencing of the transcriptome of C. chloroleuca following mycoparasitization of the sclerotia of Sclerotinia sclerotiorum revealed significant upregulation of a mitogen-activated protein kinase (MAPK)-encoding gene, crmapk. Although MAPKs are known to regulate fungal growth and development, the function of crmapk in C. chloroleuca mycoparasitism is unclear. In this study, we investigated the role of crmapk in C. chloroleuca mycoparasitism through gene knockout and complementation. Deletion of crmapk had no influence on the C. chloroleuca morphological characteristics but could significantly reduce the mycoparasitic ability to sclerotia and biocontrol capacity to soybean Sclerotinia stem rot; crmapk complementation restored these abilities. Transcriptome analysis between Δcrmapk and the wild-type strain revealed numerous genes were significantly down-regulated after crmapk deletion, including cytochrome P450, transporters, and cell wall-degrading enzymes (CWDEs). Our findings indicate that crmapk influences C. chloroleuca mycoparasitism by regulation of genes controlling the activity of CWDEs or antibiotic production. This study provides a basis for further studies of the molecular mechanism of C. chloroleuca mycoparasitism.

The heat shock protein 70 gene is involved for colony morphology, sporulation and mycoparasitism of Clonostachys rosea.

Heat shock protein 70 (HSP70) is an evolutionarily conserved chaperone protein. However, the role of HSP70 in mycoparasitism is unclear. Clonostachys rosea shows great potential against plant fungal pathogens. An HSP70 encoding gene, crhsp, from C. rosea 67-1 was significantly upregulated during C. rosea parasitization of the sclerotia of Sclerotinia sclerotiorum. In the present study, we investigated the role of crhsp in mycoparasitism using gene knockout experiments. The results showed that disruption of crhsp had remarkabe effects on the morphological characteristics of C. rosea. In addition, the ability of C. rosea to parasitize sclerotia and control soybean Sclerotinia stem rot in the greenhouse was significantly reduced in the Δcrhsp mutant. The results indicated that crhsp is involved in C. rosea mycoparasitism and provide the basis for further study of the molecular mechanism of C. rosea mycoparasitism. This is the first report to demonstrate the involvement of the HSP70 gene in C. rosea mycoparasitism.

Transcriptome analysis of virulence-differentiated Fusarium oxysporum f. sp. cucumerinum isolates during cucumber colonisation reveals pathogenicity profiles.

BACKGROUND: Cucumber Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (Foc), is one of the most notorious diseases in cucumber production. Our previous research showed the virulence of Foc significantly increases over consecutive rounds of infection in a resistant cultivar. To understand the virulence variation of Foc under host pressure, the mildly virulent strain foc-3b (WT) and its virulence-enhanced variant Ra-4 (InVir) were selected and their transcriptome profiles in infected cucumber roots were analyzed at 24 h after inoculation (hai) and 120 hai. RESULTS: A series of differentially expressed genes (DEGs) potentially involved in fungal pathogenicity and pathogenicity variation were identified and prove mainly involved in metabolic, transport, oxidation-reduction, cell wall degradation, macromolecules modification, and stress and defense. Among these DEGs, 190 up- and 360 down-regulated genes were expressed in both strains, indicating their importance in Foc infection. Besides, 286 and 366 DEGs showed up-regulated expression, while 492 and 214 showed down-regulated expression in InVir at 24 and 120 hai, respectively. These DEGs may be involved in increased virulence. Notably, transposases were more active in InVir than WT, indicating transposons may contribute to adaptive evolution. CONCLUSIONS: By a comparative transcriptome analysis of the mildly and highly virulent strains of Foc during infection of cucumber, a series of DEGs were identified that may be associated with virulence. Hence, this study provides new insight into the transcriptomic profile underlying pathogenicity and virulence differentiation of Foc.

Identification of genes related to chlamydospore formation in Clonostachys rosea 67-1.

Chlamydospores are specific structures that are of great significance to the commercialization of fungal biopesticides. To explore the genes associated with chlamydospore formation, a biocontrol fungus Clonostachys rosea 67-1 that is capable of producing resistant spores under particular conditions was investigated by transcriptome sequencing and analysis. A total of 549,661,174 clean reads were obtained, and a series of differentially expressed genes potentially involved in fungal chlamydospore formation were identified. At 36 hr, 67 and 117 genes were up- and downregulated in C. rosea during chlamydospore production, compared with the control for conidiation, and 53 and 24 genes were up- and downregulated at 72 hr. GO classification suggested that the differentially expressed genes were related to cellular component, biological process, and molecular function categories. A total of 188 metabolism pathways were linked to chlamydospore production by KEGG analysis. Sixteen differentially expressed genes were verified by reverse transcription quantitative PCR, and the expression profiles were consistent with the transcriptome data. To the best of our knowledge, it is the first report on the genes associated with chlamydospore formation in C. rosea. The results provide insight into the molecular mechanisms underlying C. rosea sporulation, which will assist the development of fungal biocontrol agents.

Out in the Cold: Identification of Genomic Regions Associated With Cold Tolerance in the Biocontrol Fungus Clonostachys rosea Through Genome-Wide Association Mapping.

There is an increasing importance for using biocontrol agents in combating plant diseases sustainably and in the long term. As large scale genomic sequencing becomes economically viable, the impact of single nucleotide polymorphisms (SNPs) on biocontrol-associated phenotypes can be easily studied across entire genomes of fungal populations. Here, we improved a previously reported genome assembly of the biocontrol fungus Clonostachys rosea strain IK726 using the PacBio sequencing platform, which resulted in a total genome size of 70.7 Mbp and 21,246 predicted genes. We further performed whole-genome re-sequencing of 52 additional C. rosea strains isolated globally using Illumina sequencing technology, in order to perform genome-wide association studies in conditions relevant for biocontrol activity. One such condition is the ability to grow at lower temperatures commonly encountered in cryic or frigid soils in temperate regions, as these will be prevalent for protecting growing crops in temperate climates. Growth rates at 10°C on potato dextrose agar of the 53 sequenced strains of C. rosea were measured and ranged between 0.066 and 0.413 mm/day. Performing a genome wide association study, a total of 1,478 SNP markers were significantly associated with the trait and located in 227 scaffolds, within or close to (< 1000 bp distance) 265 different genes. The predicted gene products included several chaperone proteins, membrane transporters, lipases, and proteins involved in chitin metabolism with possible roles in cold tolerance. The data reported in this study provides a foundation for future investigations into the genetic basis for cold tolerance in fungi, with important implications for biocontrol.

The transcription factor-encoding gene crtf is involved in Clonostachys chloroleuca mycoparasitism on Sclerotinia sclerotiorum.

Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a potential biocontrol fungus active against various fungal plant pathogens. From transcriptome sequencing of 67-1 parasitizing sclerotia of Sclerotinia sclerotiorum, we identified the transcription factor-encoding gene crtf that is significantly up-regulated during mycoparasitism. Transcription factors are widely distributed in fungi and involved in multiple biological processes. However, their role and regulatory mechanisms in mycoparasitism remain poorly understood. In this study, the function of crtf during 67-1 mycoparasitism was verified through gene knockout and complementation. The results showed that deletion of crtf did not influence fungal morphological characteristics, but the ability of the Δcrtf mutant to parasitize sclerotia and suppress soybean Sclerotinia white mold in the greenhouse was markedly diminished compared with the wild type strain. The biocontrol activity of Δcrtf recovered wild type levels when complemented with a plasmid expressing the crtf gene. These findings suggest that crtf plays a crucial role in C. chloroleuca mycoparasitism and provide insight into the molecular mechanisms underlying C. chloroleuca mycoparasitism on plant pathogenic fungi.

Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum.

Clonostachys rosea is a promising biocontrol fungus active against various plant fungal pathogens. In this study, the endochitinase-encoding gene Chi67-1, the expression of which is sharply upregulated in C. rosea 67-1 when induced by sclerotia, was transformed into the original isolate by protoplast transformation, and transformants were screened against Sclerotinia rot of soybean. The transformation efficiency was approximately 50 transformants per 1 × 107 protoplasts, and 68 stably heritable recombinants were assayed. The parasitic rates of 32.4% of the tested strains increased by more than 50% compared to 43.3% of the wild type strain in 16 h, and the Rc4-4 transformant showed a parasitic rate of 100% in 16 h. The control efficiencies of the selected efficient transformants to soybean Sclerotinia stem rot were evaluated in pots in the greenhouse, and the results revealed that Rc4-4 achieved the highest efficiency of 81.4%, which was 31.7% and 28.7% higher than the control achieved by the wide type and the pesticide carbendazim, respectively. Furthermore, the expression level of Chi67-1 was 107-fold higher in Rc4-4 than in the wild type, and accordingly, the chitinase activity of the recombinant increased by 140%. The results lay a foundation for the development of efficient genetically engineered strains of C. rosea.

Transaldolase gene Tal67 enhances the biocontrol activity of Clonostachys rosea 67-1 against Sclerotinia sclerotiorum.

Clonostachys rosea is a promising biocontrol agent that parasitizes various fungal plant pathogens. In this paper, transaldolase gene Tal67 was found to be greatly upregulated in C. rosea isolate 67-1 during mycoparasitism of Sclerotinia sclerotiorum sclerotia. Quantitative real-time PCR revealed a significant increase in expression at 0-48 h after induction by sclerotia, and the level peaked at 13.9-fold higher than the control at 24 h. Gene disruption led to a decrease in the growth rate of the Tal67-deficient strain ΔTal67 to 5.3 mm/day, which was much lower than the wild type and the complemented strain ΔTal67+ (P < 0.05). The antagonistic activity of ΔTal67 against Botrytis cinerea was 15.8% lower than the wild type, and the parasitic rate to S. sclerotiorum decreased by 24.6%. However, reinsertion of the transaldolase gene recovered the fungicidal activity of C. rosea. The efficacy of the mutants against soybean Sclerotinia stem rot was evaluated in the greenhouse, and the control efficiency of isolate 67-1 reached 65.3%, while the efficiency of the ΔTal67 strain decreased sharply to 17.8%, and the complemented strain ΔTal67+ recovered to 64.8%. These results suggest that Tal67 plays an important role in the growth and biocontrol activity of C. rosea.

Identification of mycoparasitism-related genes in Clonostachys rosea 67-1 active against Sclerotinia sclerotiorum.

Clonostachys rosea is a mycoparasite that has shown great potential in controlling various plant fungal pathogens. In order to find mycoparasitism-related genes in C. rosea, the transcriptome of the efficient isolate 67-1 in association with sclerotia of Sclerotinia sclerotiorum was sequenced and analysed. The results identified 26,351 unigenes with a mean length of 1,102 nucleotides, among which 18,525 were annotated in one or more databases of NR, KEGG, Swiss-Prot, GO and COG. Differentially expressed genes at 8 h, 24 h and 48 h after sclerotial induction were analysed, and 6,890 unigenes were upregulated compared with the control without sclerotia. 713, 1,008 and 1,929 genes were specifically upregulated expressed, while 1,646, 283 and 529 genes were specifically downregulated, respectively. Gene ontology terms analysis indicated that these genes were mainly involved in metabolism of biological process, catalysis of molecular function and cellular component. The expression levels of 12 genes that were upregulated after encountering with S. sclerotiorum were monitored using real-time PCR. The results indicated that the quantitative detection was consistent with the transcriptome analysis. The study provides transcriptional gene expression information on C. rosea parasitizing S. sclerotiorum and forms the basis for further investigation of mycoparasitism-related genes of C. rosea.

Draft Genome Sequence of Mycoparasite Clonostachys rosea Strain 67-1.

Clonostachys rosea is a promising mycoparasite. In this study, we sequenced the draft genome of the highly effective strain 67-1 using the Illumina HiSeq 2500 sequencing platform. The genome is 55.4 Mb with a G+C content of 49.2% and provides a powerful resource for future studies on the molecular mechanisms underlying Clonostachys rosea's antagonism on fungal pathogens.

Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction.

Reference genes are important to precisely quantify gene expression by real-time PCR. In order to identify stable and reliable expressed genes in mycoparasite Clonostachys rosea in different modes of nutrition, seven commonly used housekeeping genes, 18S rRNA, actin, ß-tubulin, elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme and glyceraldehyde-3-phosphate dehydrogenase, from the effective biocontrol isolate C. rosea 67-1 were tested for their expression under sclerotial induction and during vegetative growth on PDA medium. Analysis by three software programs showed that differences existed among the candidates. Elongation factor 1 was most stable; the M value in geNorm, SD value in Bestkeeper and stability value in Normfinder analysis were 0.405, 0.450 and 0.442, respectively, indicating that the gene elongation factor 1 could be used to normalize gene expression in C. rosea in both vegetative growth and parasitic process. By using elongation factor 1, the expression of a serine protease gene, sep, in different conditions was assessed, which was consistent with the transcriptomic data. This research provides an effective method to quantitate expression changes of target genes in C. rosea, and will assist in further investigation of parasitism-related genes of this fungus.

A perilipin gene from Clonostachys rosea f. Catenulata HL-1-1 is related to sclerotial parasitism.

Clonostachys rosea f. catenulata is a promising biocontrol agent against many fungal plant pathogens. To identify mycoparasitism-related genes from C. rosea f. catenulata, a suppression subtractive hybridization (SSH) cDNA library of C. rosea f. catenulata HL-1-1 that parasitizes the sclerotia of S. sclerotiorum was constructed. 502 clones were sequenced randomly, and thereby 472 expressed sequence tags (ESTs) were identified. Forty-three unigenes were annotated and exhibited similarity to a wide diversity of genes. Quantitative real-time PCR showed that a perilipin-like protein encoding gene, Per3, was up-regulated by 6.6-fold over the control at 96 h under the induction of sclerotia. The full-length sequence of Per3 was obtained via 5' and 3' rapid identification of cDNA ends. Overexpression of Per3 in HL-1-1 significantly enhanced the parasitic ability on sclerotia. The results indicated that Per3 might be involved in the mycoparasitism of C. rosea f. catenulata HL-1-1. This is the first report of a perilipin as a potential biocontrol gene in mycoparasites. The study provides usefu l insights into the interaction between C. rosea f. catenulata and fungal plant pathogens.

Synergistic Effect of Dazomet Soil Fumigation and Clonostachys rosea Against Cucumber Fusarium Wilt.

Soil fumigation and biological control are two control measures frequently used against soilborne diseases. In this study, the chemical fumigant dazomet was applied in combination with the biocontrol agent (BCA) Clonostachys rosea 67-1 to combat cucumber wilt caused by Fusarium oxysporum f. sp. cucumerinum KW2-1. When the mycoparasite C. rosea 67-1 was applied after dazomet fumigation, disease control reached 100%, compared with 88.1 and 69.8% for dazomet and 67-1 agent, respectively, applied alone, indicating a synergistic effect of dazomet and C. rosea in combating cucumber Fusarium wilt based on analysis of Bliss Independence. To understand the synergistic mechanism, the effects of chemical fumigation on the colonization potential and activity of F. oxysporum f. sp. cucumerinum, and the interaction between the BCA and the pathogen were investigated. The results showed that growth of the pathogen decreased with increasing dazomet concentration subsequent to fumigation. When exposed to dazomet at 100 ppm, the fungal sporulation rate decreased by 94.4%. Severe damage was observed in fumigated isolates using scanning electron microscopy. In the greenhouse, disease incidence of cucumber caused by fumigated F. oxysporum f. sp. cucumerinum significantly decreased. Whereas germination of C. rosea 67-1 spores increased by >sixfold in fumigated soil, and its ability to parasitize fumigated F. oxysporum f. sp. cucumerinum significantly increased (P = 0.014).

[Diversity of actinomycetes associated with root-knot nematode and their potential for nematode control].

Twenty actinomycetes were isolated from root-knot nematode eggs and females collected from 11 plant root samples infested by Meloidogyne spp.. The isolates were assigned to the genera Streptomyces, Nocardia and Pseudonocardia respectively, based on analysis of morphological characteristics, cell-wall DAPs and 16S rRNA gene sequences. 80% of them were streptomycetes. Biocontrol potential of the isolates against Meloidogyne hapla was evaluated in liquid culture in vitro. The average percentages of egg parasitism, egg hatching, and juvenile mortality were 54.1, 40.4 and 26.2, respectively. Three Streptomyces strains and one Nocardia strain with high pathogenicity in vitro were selected to determine their ability to reduce tomato root galls in greenhouse. The results demonstrated good biocontrol efficacy (31.4%-56.4%) of the strains.

Fungi and actinomycetes associated with Meloidogyne spp. eggs and females in China and their biocontrol potential.

A survey was conducted to determine the microflora on eggs and females of Meloidogyne spp. collected from plant roots and infested soil in China. A total of 455 fungal isolates belonging to 24 genera and 52 isolates of actinomycetes were obtained from 28 samples from greenhouses and fields in Hainan, Yunnan, Fujian, Hebei, Shandong, and Beijing. The predominant fungal species were Paecilomyces lilacinus (49.3% of the isolates), Fusarium spp. (7.9%), Pochonia chlamydosporia (6.9%), Penicillium spp. (5.7%), Aspergillus spp. (3.2%), and Acremonium spp. (2.8%). Actinomycetes were frequently encountered (10.3%) as well. A total of 350 isolates of nematophagous fungi and actinomycetes were evaluated for their parasitism of eggs and effects on egg hatch and juvenile mortality in vitro. Pathogenicity varied among isolates, and 29.1% of isolates parasitized over 90% eggs 4 days after inoculation. Results also show that seven isolates of fungi and actinomycetes reduced egg hatch rates to less than 10% contrasted to the control of 65.8%, and three isolates killed all hatched juveniles after 7 days. Seventeen fungal isolates and four actinomycete isolates with high pathogenicity in vitro were selected to test biocontrol efficacy in the greenhouse. They reduced tomato root gall index by 13.4-58.9% compared to the no treatment control.

The Analysis of Loudness Discomfort Level and Hyperacusis in Tinnitus Patients / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Hyperacusis is an abnormally strong reaction which occurs within auditory pathways resulting from exposure to moderate sound. More patients are inflicted with it than with tinnitus, accounting for a relatively high proportion of the total tinnitus patients. However, no single test could pass as an objective diagnostic test of hyperacusis. Among various psychoacoustic evaluations, loudness discomfort level (LDL) is frequently used in the evaluation of hyperacusis. Thus, this study aimed to demonstrate a proportion of hyperacusis in tinnitus patients and characterize LDL in hyperacusis and tinnitus patients as a diagnostic test. MATERIALS AND METHOD: Using 186 patients with tinnitus as experiment group and 35 acoustically normal persons as control group, pure tone audiometry (PTA), LDL, and dynamic range (DR) were checked. RESULTS: Amongst the total number of tinnitus patients, there were 10 hyperacusis patients (5.38%). Those hyperacusis patients had much lower LDL (73.50 dB) and DR (41.50 dB) than tinnitus patients without hyperacusis (LDL: 100.90 dB, DR: 67.20 dB) as well as the control group (LDL: 104.86 dB, DR: 93.98 dB). CONCLUSION: Hyperacusis patients consist 5.38% (10/186) and have much lower LDL than tinnitus patients without hyperacusis and the control group. There is a positive correlation between hyperacusis and LDL. Therefore, in a diagnosis of hyperacusis, it is essential to check LDL and take a careful look into the history from patients.

Clinical Study of Frontal Sinus Fractures: A Review of 14 Cases / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The incidence of frontal sinus fractures has increased as traffic and industrial accidents have increased. However, they are still uncommon compared to other facial traumas because of the arch configuration and the thickness of the anterior wall of the frontal sinus. Accurate evaluation is highly recommended in cases of frontal sinus fractures because they are likely to result in serious complications, including frontal sinusitis, meningitis, and mucocele. In this paper, we reviewed 14 cases of frontal sinus fractures and investigated the clinical characteristics and treatment results. MATERIALS AND METHODS: A retrospective review was performed on 14 patients with frontal sinus fractures who had been treated in our department from June, 1995 to August, 1997. RESULTS: Eleven patients were treated with surgery, including osteoplastic frontal sinus surgery with fat obliteration (5 patients), open reduction and internal fixation of the anterior wall (4 patients), and cranialization (2 patients). Three patients were treated with conservative methods. There were no complications such as infection, meningitis, and mucocele formation in the patients who underwent surgery for an average follow-up period of 19 months. CONCLUSION: Our results show that an aggressive approach is justified for treatment of frontal sinus fractures.