Kehe Fu

Trichoderma biodiversity in China

In the present study, we made further investigation into the diversity of Trichoderma in China than previous ones utilizing comprehensive approaches of morphological microscopic observation and phylogenetic analysis by detecting molecular markers. One thousand nine hundred ten Trichoderma strains were isolated from soil or other materials in China: East (Anhui, Fujian, Jiangsu, Jiangxi, Shandong, Zhejiang province and Shanghai municipality), South-West (Guizhou, Qinghai, Shanxi, Sichuan and Yunnan province, Tibet Autonomous Region and Chongqing municipality), South-East (Guangdong, Guangxi, Hainan province), and Middle China (Henan, Hubei and Hunan province). Representative isolates were verified at the species level by morphological characters and the oligonucleotide barcode program TrichoOKey v.10 and the custom BLAST server TrichoBLAST, using sequence of the ITS 1 and 2 region of the rDNA cluster and partial sequences of translation elongation factor 1-alpha(tef1-α). A total of 23 Trichoderma species were identified : T.asperellum, T.atrioviride, T.aureovriride, T.brevicompactum, T.citrioviride, T.erinaceum, T.gamsii, T.hamatum, T.harzianum (H.1ixii), T.intricatum, T.koningii (H.koningii), T.koningiopsis, T.longibranchiatum, T.pleuroticola, T.reeseii (H.jecorina), T.sinensis, T.spirale, T.stromaticum, T.tomentosum, T.velutinum, T.vermipilum, T.virens (H.virens), T.viride. Among them, 3 species: T.intricatum, T.stromaticum, T.vermipilum were first reported in China; T.harzianum (H,1ixii) was the most widely distributed species in China. This study further shows that, the highest biodiversity of Trichoderma population appeared in South-West China.

Genome sequence and virulence variation-related transcriptome profiles of Curvularia lunata, an important maize pathogenic fungus

BackgroundCurvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China. Genome sequencing of the pathogen will provide important information for globally understanding its virulence mechanism.ResultsWe report the genome sequences of a highly virulent C. lunata strain. Phylogenomic analysis indicates that C. lunata was evolved from Bipolaris maydis (Cochliobolus heterostrophus). The highly virulent strain has a high potential to evolve into other pathogenic stains based on analyses on transposases and repeat-induced point mutations. C. lunata has a smaller proportion of secreted proteins as well as B. maydis than entomopathogenic fungi. C. lunata and B. maydis have a similar proportion of protein-encoding genes highly homologous to experimentally proven pathogenic genes from pathogen-host interaction database. However, relative to B. maydis, C. lunata possesses not only many expanded protein families including MFS transporters, G-protein coupled receptors, protein kinases and proteases for transport, signal transduction or degradation, but also many contracted families including cytochrome P450, lipases, glycoside hydrolases and polyketide synthases for detoxification, hydrolysis or secondary metabolites biosynthesis, which are expected to be crucial for the fungal survival in varied stress environments. Comparative transcriptome analysis between a lowly virulent C. lunata strain and its virulence-increased variant induced by resistant host selection reveals that the virulence increase of the pathogen is related to pathways of toxin and melanin biosynthesis in stress environments, and that the two pathways probably have some overlaps.ConclusionsThe data will facilitate a full revelation of pathogenic mechanism and a better understanding of virulence differentiation of C. lunata.

Understanding resistant germplasm-induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata.

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up‐regulated and four down‐regulated proteins were identified in virulence‐increased strain compared to WS18. Our analysis revealed that melanin synthesis‐related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance‐related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence‐increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata.

Biological role of Trichoderma harzianum-derived platelet-activating factor acetylhydrolase (PAF-AH) on stress response and antagonism.

We investigated the properties of platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum. The enzyme, comprised of 572 amino acids, shares high homology with PAF-AH proteins from T. koningii and other microbial species. The optimum enzymatic activity of PAF-AH occurred at pH 6 in the absence of Ca2+ and it localized in the cytoplasm, and we observed the upregulation of PAF-AH expression in response to carbon starvation and strong heat shock. Furthermore, PAF-AH knockout transformant growth occurred more slowly than wild type cells and over-expression strains grown in SM medium at 37°C and 42°C. In addition, PAF-AH expression significantly increased under a series of maize root induction assay. Eicosanoic acid and ergosterol levels decreased in the PAF-AH knockouts compared to wild type cells, as revealed by GC/MS analysis. We also determined stress responses mediated by PAF-AH were related to proteins HEX1, Cu/Zn superoxide dismutase, and cytochrome c. Finally, PAF-AH exhibited antagonistic activity against Rhizoctonia solani in plate confrontation assays. Our results indicate PAF-AH may play an important role in T. harzianum stress response and antagonism under diverse environmental conditions.

Cellulase from Trichoderma harzianum interacts with roots and triggers induced systemic resistance to foliar disease in maize.

Trichoderma harzianum is well known to exhibit induced systemic resistance (ISR) to Curvularia leaf spot. We previously reported that a C6 zinc finger protein (Thc6) is responsible for a major contribution to the ISR to the leaf disease, but the types of effectors and the signals mediated by Thc6 from Trichoderma are unclear. In this work, we demonstrated that two hydrolases, Thph1 and Thph2, from T. harzianum were regulated by Thc6. Furthermore, an electrophoretic mobility shift assay (EMSA) study revealed that Thc6 regulated mRNA expression by binding to GGCTAA and GGCTAAA in the promoters of the Thph1 and Thph2 genes, respectively. Moreover, the Thph1 and Thph2 proteins triggered the transient production of reactive oxygen species (ROS) and elevated the free cytosolic calcium levels in maize leaf. Furthermore, the genes related to the jasmonate/ethylene signaling pathway were up-regulated in the wild-type maize strain. However, the ΔThph1- or ΔThph2-deletion mutants could not activate the immune defense-related genes in maize to protect against leaf disease. Therefore, we conclude that functional Thph1 and Thph2 may be required in T. harzianum to activate ISR in maize.

Construction of a Streptomyces lydicus A01 transformant with a chit42 gene from Trichoderma harzianum P1 and evaluation of its biocontrol activity against Botrytis cinerea

Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.

Tmac1, a transcription factor which regulated high affinity copper transport in Trichoderma reesei

The Mac1 protein was a transcriptional activator that sensed very low concentration of copper and regulated the copper transport in Saccharomyces cerevisiae. Here, we cloned a gene from Trichoderma reesei named Tmac1, whose deduced amino acid sequence showed 29% identical to Mac1p. Furthermore, two Cys-His repeats metal binding motifs of Tmac1p, one in the 354-369C terminus and one in the 475-490C terminus were also present in Mac1p. A deletion mutant of Tmac1 was hypersensitive to the copper starvation and showed poor growth. Subsequently, the function was recovered by the gene complementation experiment. Furthermore, the Tmac1 gene fully complemented growth defects of yeast ΔMac1 mutant. The expression of Tmac1p was activated at low concentration of copper and depressed when the concentration of copper excess 1mM. Furthermore, the fluorescence intensity enhanced at copper starvation and decreased under copper excess by fusion the eGFP to the Tmac1p. It proved that the expression of Mac1p was exactly regulated by copper concentration, because eGFP and Mac1p were expressed under the control of the same one promoter. We also cloned a gene named Tctr3 with bioinformatics. With a series of experiments, we proved it was the target gene of Tmac1. To sum up, Tmac1 may encode a transcriptional activator regulated high-affinity copper transport in T. reesei.

Thc6 protein, isolated from Trichoderma harzianum, can induce maize defense response against Curvularia lunata

Mutant T66 was isolated from 450 mutants (constructed with Agrobacterium tumefaciens‐mediated transformation method) of Trichoderma harzianum. Maize seeds coated with T66 were more susceptible to Curvularia lunata when compared with those coated with wild‐type (WT) strain. The disease index of maize treated with T66 and WT were 62.5 and 42.1%, respectively. Further research showed T‐DNA has inserted into the ORF of one gene, which resulted in the functional difference between WT and T66. The gene was cloned and named Thc6, which encodes a novel 327 amino acid protein. To investigate its function, we obtained knockout, complementation, and overexpression mutants of Thc6. Challenge inoculation studies suggested that the Thc6 overexpression mutant can reduce the disease index of maize inbred line Huangzao 4 against the leaf spot pathogen (C. lunata). Meanwhile, The Thc6 mutants were found to affect the resistance of maize inbred line Huangzao 4 against C. lunata by enhancing the activation of jasmonate‐responsive genes expression. Liquid chromatography–mass spectrometry (LC–MS) data further confirmed that the concentration of jasmonate in the induced maize exhibits a parallel change tendency with the expression level of defense‐related genes. Hence, the Thc6 gene could be participated in the induced resistance of maize inbred line Huangzao 4 against C. lunata infection through a jasmonic acid‐dependent pathway.

Increased virulence of transgenic Trichoderma koningi strains to the Asian corn borer larvae by overexpressing heterologous chit42 gene with chitin-binding domains

The chit42 gene cloned from Metarhizium anisopliae lacks chitin-binding domain (chBD), which plays important roles in binding insoluble chitin. Five kinds of hybrid chitinase Trichoderma transformants were constructed in this study, where the chit42 gene was fused to chBDs derived from plant, bacterial, and insect sources. The transformant Mc4 harboring chBDs from bitter melon (Momordica charantia) displayed the highest chitinase activity among all chBDs. The chitinase activities of Mc4, chit42 Trichoderma transformant Mchit3, and wild-type strain T30 were 44.94, 32.48, and 12.38 U/mL, respectively. The mortality rate of corn borer larvae in Mc4 fermentation liquid treatment increased by 10% and 30% compared with Mchit3 and T30, respectively. The midgut microvilli and goblet cell microvilli of the corn borer larvae exhibited distinct pathological changes after 48 h of feeding in Mc4 treatment. Mc4 also exhibited the strongest antifungal activity against Fusarium verticillioides and Rhizoctonia solani.

Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis) by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon–Wiener (H) and the Simpson (1-D) indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.