The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

The Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant.

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.

Candidate Effectors from Botryosphaeria dothidea Suppress Plant Immunity and Contribute to Virulence.

Fungal effectors play important roles in host-pathogen interactions. Botryosphaeria dothidea is an ascomycetous fungus that is responsible for the diseases of hundreds of woody plant species, including apple ring rot, which seriously affects apples worldwide. However, little is known about the effectors of B. dothidea. In this study, we analyzed the B. dothidea genome and predicted 320 candidate effector genes, 124 of which were successfully amplified and cloned. We investigated the effects of these genes on plant cell death in Nicotiana benthamiana while using a transient expression system. Twenty-four hours after initial inoculation with Agrobacterium tumefaciens cells carrying candidate effectors, the infiltrated leaves were challenged with A. tumefaciens cells carrying the BAX gene. In total, 116 candidate effectors completely inhibited, while one partially inhibited, the programmed cell death (PCD) of N. benthamiana induced by BAX, whereas seven candidate effectors had no effect. We then further tested seven candidate effectors able to suppress BAX-triggered PCD (BT-PCD) and found that they all completely inhibited PCD triggered by the elicitors INF1, MKK1, and NPK1. This result suggests that these effectors were activated in order to suppress pathogen-associated molecular pattern-triggered immunity. The signal peptides of these candidate effectors exhibited secretory activity in yeast (pSUC2 vector). Moreover, the respective deletion of Bdo_11198 and Bdo_12090 significantly reduced the virulence of B. dothidea. These results suggest that these effectors play important roles in the interaction of B. dothidea with its hosts.

An efficient gene disruption method for the woody plant pathogen Botryosphaeria dothidea.

BACKGROUND: Botryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea. RESULTS: We established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 106 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 µg per 106 protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red. CONCLUSION: The established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.

Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit.

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Characterization of a novel strain of Botryosphaeria dothidea chrysovirus 1 from the apple white rot pathogen Botryosphaeria dothidea.

A novel strain of Botryosphaeria dothidea chrysovirus 1 was identified. It encodes a shortened RNA-dependent RNA polymerase and an elongated coat protein, and it might cause hypovirulence of the host fungal strain.

Diversity, evolution and expression profiles of histone acetyltransferases and deacetylases in oomycetes.

BACKGROUND: Oomycetes are a group of fungus-like eukaryotes with diverse microorganisms living in marine, freshwater and terrestrial environments. Many of them are important pathogens of plants and animals, causing severe economic losses. Based on previous study, gene expression in eukaryotic cells is regulated by epigenetic mechanisms such as DNA methylation and histone modification. However, little is known about epigenetic mechanisms of oomycetes. RESULTS: In this study, we investigated the candidate genes in regulating histone acetylation in oomycetes genomes through bioinformatics approaches and identified a group of diverse histone acetyltransferases (HATs) and histone deacetylases (HDACs), along with three putative novel HATs. Phylogenetic analyses suggested that most of these oomycetes HATs and HDACs derived from distinct evolutionary ancestors. Phylogenetic based analysis revealed the complex and distinct patterns of duplications and losses of HATs and HDACs in oomycetes. Moreover, gene expression analysis unveiled the specific expression patterns of the 33 HATs and 11 HDACs of Phytophthora infestans during the stages of development, infection and stress response. CONCLUSIONS: In this study, we reveal the structure, diversity and the phylogeny of HATs and HDACs of oomycetes. By analyzing the expression data, we provide an overview of the specific biological stages of these genes involved. Our datasets provide useful inputs to help explore the epigenetic mechanisms and the relationship between genomes and phenotypes of oomycetes.

Monilinia Species Associated with Brown Rot of Cultivated Apple and Pear Fruit in China.

Monilinia isolates were collected from major apple and pear production regions in China from 2004 to 2011 and identified based on their morphological characteristics and three highly conserved loci. The 247 isolates belonged to three species: Monilinia fructicola, Monilia yunnanensis, and Monilia polystroma. M. yunnanensis was the most prevalent (77%), followed by M. polystroma (20%) and Monilinia fructicola (3%). Monilia yunnanensis is primarily distributed in the south, north, and west of China; M. polystroma is limited to the north and east; and Monilinia fructicola was detected only from a few samples from the north and east. Phylogenetic analysis based on internal transcribed spacer, ß-tubulin, and laccase (lcc2) genes suggested that Monilia yunnanensis, M. polystroma, and Monilinia fructigena are closely related, and Monilia yunnanensis is more distantly related. We also found that these three species do not show consistent differences in morphological characteristics, including colony morphology, colony expansion rate, conidial characteristics, and the amount of stroma produced in culture. Thus, these three species are more like phylogenetic species in the process of speciation. In addition, a set of species-specific primers based on single-nucleotide polymorphisms and deletions in the lcc2 gene region were designed and a conventional polymerase chain reaction method successfully developed for differentiating Monilinia fructicola, Monilia yunnanensis, M. polystroma, and Monilinia laxa from the other species.

The Arabidopsis lectin receptor kinase LecRK-I.9 enhances resistance to Phytophthora infestans in Solanaceous plants.

Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.

Methylation entropy landscape of Chinese long-lived individuals reveals lower epigenetic noise related to human healthy aging.

The transition from ordered to noisy is a significant epigenetic signature of aging and age-related disease. As a paradigm of healthy human aging and longevity, long-lived individuals (LLI, >90 years old) may possess characteristic strategies in coping with the disordered epigenetic regulation. In this study, we constructed high-resolution blood epigenetic noise landscapes for this cohort by a methylation entropy (ME) method using whole genome bisulfite sequencing (WGBS). Although a universal increase in global ME occurred with chronological age in general control samples, this trend was suppressed in LLIs. Importantly, we identified 38,923 genomic regions with LLI-specific lower ME (LLI-specific lower entropy regions, for short, LLI-specific LERs). These regions were overrepresented in promoters, which likely function in transcriptional noise suppression. Genes associated with LLI-specific LERs have a considerable impact on SNP-based heritability of some aging-related disorders (e.g., asthma and stroke). Furthermore, neutrophil was identified as the primary cell type sustaining LLI-specific LERs. Our results highlight the stability of epigenetic order in promoters of genes involved with aging and age-related disorders within LLI epigenomes. This unique epigenetic feature reveals a previously unknown role of epigenetic order maintenance in specific genomic regions of LLIs, which helps open a new avenue on the epigenetic regulation mechanism in human healthy aging and longevity.

Phytophthora infestans field isolates from Gansu province, China are genetically highly diverse and show a high frequency of self fertility.

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self-fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within-area and among-area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Population Structure of Brown Rot Fungi on Stone Fruits in China.

In total, 455 Monilinia isolates from stone fruits collected from several provinces (cities) in China from 2003 to 2009 were identified to species based on morphological characteristics, molecular identification, and the sequence of the internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of the ribosomal RNA. Overall, four species were detected (Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma). M. fructicola was the most prevalent (93.0%) followed by M. fructigena (4.8%), M. laxa (2.0%), and Monilia polystroma (0.2%). M. fructicola and M. fructigena were found on peach, plum, and apricot; M. laxa was found only on apricot, cherry (in an organic orchard), and wild peach; and Monilia polystroma was found only on plum in Heilongjiang. The pathogenicity of Monilinia fructicola, M. laxa, and M. fructigena did not significantly differ on wounded nectarine and apricot, indicating that the differences in frequency of occurrence were not linked to virulence. Phylogenetic analysis based on ITS sequences showed that the isolates of M. laxa and M. fructigena from China differed from isolates of these species from other countries, and that the difference led to the separation of the isolates from China and those from other countries into different phylogenetic groups. Further study is needed to determine whether they are cryptic species.

Photoredox-Catalyzed Difunctionalization of Unactivated Olefins for Synthesizing Lactam-Substituted gem-Difluoroalkenes.

Herein, the synthesis of lactam-substituted gem-difluoroalkenes has been developed through a photoredox-catalyzed radical cascade reaction. This developed photoredox-catalyzed, Brønsted base-assisted intramolecular 5-exo-trig cyclization/intermolecular radical addition/ß-fluoride elimination reaction offers a simple method for producing lactam, carbamate, or urea-substituted gem-difluoroalkenes with good functional group tolerance and high yields.

Genetic diversity of populations of Monilinia fructicola (Fungi, Ascomycota, Helotiales) from China.

The genetic variation among 128 isolates of Monilinia fructicola (Fungi, Ascomycota, Helotiales) from China was analyzed using Inter-Simple Sequence Repeat (ISSR) markers and compared with those of samples from California, USA and New Zealand. A total of 72 reproducible DNA fragments were scored, of which 87.5% (63/72) were polymorphic. The Nei's gene diversity and Shannon's diversity indices of three Chinese regional populations were very similar to that from California. However, several differences were observed among geographic populations of M. fructicola from both within China and between China and California. The analysis of molecular variance (AMOVA) of isolates from different geographic locations suggested that most of the observed genetic variation was found within populations. Results of this study are inconsistent with the hypothesis that the Chinese populations of M. fructicola were derived from a single or few recent migrants from other countries. Instead, our results suggest that M. fructicola has been in China long before its first official recording in 2003.

Comparative Transcriptome Analysis Revealed Genes Regulated by Histone Acetylation and Genes Related to Sex Hormone Biosynthesis in Phytophthora infestans.

Late blight caused by Phytophthora infestans, is one of the most devastating diseases of potato, and was responsible for the death of millions of people during the Irish Potato Famine in the nineteenth century. Phytophthora infestans is a heterothallic oomycete that typically requires two compatible types (mating types), A1 and A2, to complete sexual reproduction (i.e., oospore production). Oospores have critical effects on disease epidemiology because they serve as the primary inoculum in subsequent growing seasons. The sexual reproduction of Phytophthora species is regulated by α hormones. In previous studies, we proved that transformants in which selected histone deacetylase (HDAC) genes are silenced exhibit abnormal hormone production. In the current study, we compared the transcriptomes of HDAC-silenced and wild-type strains to explore the genes regulated by HDAC and the genes involved in sex hormone biosynthesis in Phytophthora species. A total of 14,423 transcripts of unigenes were identified in the wild-type strain, the HDAC family-silenced transformant (HDST), and the HDAC7-silenced transformant (H7ST). After comparing the intergroup gene expression levels, 1,612 unigenes were identified as differentially expressed among these strains. The expression levels of 16 differentially expressed genes (DEGs) were validated by quantitative real-time PCR. The functional annotation of the DEGs by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that HDACs affect the expression of genes related to metabolic and biosynthetic processes, RNA processing, translation, ribosome biogenesis, cellular structural constituents, RNA binding, and protein binding. Moreover, HDAC7 specifically influences the transcription of genes associated with transport, methylation, mitochondria, organelle inner membranes, receptors and transporters, and hydrolase activities. We also identified 18 candidate genes related to α hormones biosynthesis, including a gene encoding the NF-Y transcription factor (PITG_10861). The overexpression of PITG_10861 increased the production of hormone α2. The results of this study revealed P. infestans genes affected by histone acetylation. The data presented herein provide useful inputs for future research on the epigenetic mechanisms and mating behaviors of Phytophthora species.

A proper PiCAT2 level is critical for sporulation, sporangium function, and pathogenicity of Phytophthora infestans.

Catalase is present in prokaryotic and eukaryotic organisms and is important for the protective effects of the antioxidant system against free radicals. Many studies have confirmed that catalase is required for the growth, development, and pathogenesis of bacteria, plants, animals, and fungi. However, there has been relatively little research on the catalases in oomycetes, which form an important group of fungus-like eukaryotes that produce zoosporangia. In this study, we detected two Phytophthora infestans genes encoding catalases, but only PiCAT2 exhibited catalase activity in the sporulation stage and was highly produced during asexual reproduction and in the late infection stage. Compared with the wild-type strain, the PiCAT2-silenced P. infestans transformants were more sensitive to abiotic stress, were less pathogenic, and had a lower colony expansion rate and lower PiMPK7, PiVPS1, and PiGPG1 expression levels. In contrast, the PiCAT2-overexpressed transformants were slightly less sensitive to abiotic stress. Interestingly, increasing and decreasing PiCAT2 expression from the normal level inhibited sporulation, germination, and infectivity, and down-regulated PiCdc14 expression, but up-regulated PiSDA1 expression. These results suggest that PiCAT2 is required for P. infestans mycelial growth, asexual reproduction, abiotic stress tolerance, and pathogenicity. However, a proper PiCAT2 level is critical for the formation and normal function of sporangia. Furthermore, PiCAT2 affects P. infestans sporangial formation and function, pathogenicity, and abiotic stress tolerance by regulating the expression of cell cycle-related genes (PiCdc14 and PiSDA1) and MAPK pathway genes. Our findings provide new insights into catalase functions in eukaryotic pathogens.

Identification of a novel CHN1 p.(Phe213Val) variant in a large Han Chinese family with congenital Duane retraction syndrome.

Duane retraction syndrome (DRS) is a neuromuscular dysfunction of the eyes. Although many causative genes of DRS have been identified in Europe and the United States, few reports have been published in regard to Chinese DRS. The aim of the present study was to explore the genetic defect of DRS in a Chinese family. Exome sequencing was used to identify the disease-causing gene for the two affected family members. Ophthalmic and physical examinations, as well as genetic screenings for variants in chimerin 1 (CHN1), were performed for all family members. Functional analyses of a CHN1 variant in 293T cells included a Rac-GTP activation assay, α2-chimaerin translocation assay, and co-immunoprecipitation assay. Genetic analysis revealed a NM_001822.7: c.637T > G variant in the CHN1 gene, which resulted in the substitution of a highly conserved C1 domain with valine at codon 213 (NP_001813.1: p.(Phe213Val)) (ClinVar Accession Number: SCV001335305). In-silico analysis revealed that the p.(Phe213Val) substitution affected the protein stability and connections among the amino acids of CHN1 in terms of its tertiary protein structure. Functional studies indicated that the p.(Phe213Val) substitution reduced Rac-GTP activity and enhanced membrane translocation in response to phorbol-myristoyl acetate (PMA). Together with previous studies, our present findings demonstrate that CHN1 may be an important causative gene for different ethnicities with DRS.

Phenotypic variability of SLC7A14 mutations in patients with inherited retinal dystrophy.

BACKGROUND: Inherited retinal dystrophy (IRD) is a group of retinal disorders that are both clinically and genetically diverse, typically with loss of photoreceptor function. Herein, we aimed to identify the underlying genetic defect in IRD patients with mutations in the SLC7A14 gene. METHODS: A targeted exome capture panel was applied for mutational screening of SLC7A14. Targeted exome sequencing (TES) was performed on 200 non-syndromic and unrelated autosomal recessive or sporadic IRD families. Candidate variants were validated by direct sequencing and further examined using bioinformatics analyses for determination of their potential effect. RESULTS: We identified compound heterozygous missense mutations (c.988G>A, p.G330R; c.1970G>A, p.R657Q) in an autosomal recessive retinitis pigmentosa (RP) case and a homozygous mutation (c.988G>A, p.G330R) in a simplex case with Leber congenital amaurosis (LCA) in the SLC7A14 gene. Both G330R and R657Q were deleterious based on in silico predictive tools. Our proposed topological model of the SLC7A14 polypeptide suggested that both G330R and R657Q affected evolutionarily highly conserved amino acid residues in SLC7A14 that occurred in transmembrane helixes. Structural modeling revealed a broken arginine and aspartic acid connection between residues 657 and 406. CONCLUSIONS: We applied TES to the molecular diagnosis of patients with IRD and for the first time identified SLC7A14 mutations in two unrelated families with RP and LCA separately. Our findings uniquely add the knowledge of the phenotypic variability of SLC7A14 mutations.

[Warming Needle Moxibustion Relieves Symptoms of Lumbar Disc Herniation Patients and Upregulates Plasma ß-endorphin].

OBJECTIVE: To analyze the effect of warming needle moxibustion of Jiaji (EX-B 2), Huantiao (GB 30), etc. on lumbago and other symptoms of lumbar disc herniation (LDH) patients and changes of plasma ß-endorphin (ß-EP) content. METHODS: A total of 60 LDH patients were equally randomized into warming needle moxibustion group and conventional acupuncture group. Patients of the conventional acupuncture group were treated by puncturing lumbar Jiaji (EX-B 2), Huantiao (GB 30), Weizhong (BL 40), and Yanglingquan (GB 34) with filiform acupuncture needles. Patients of the warming needle moxibustion group were treated by puncturing the same 4 acupoints, and with the acupuncture needle in lumbar EX-B 2 attached an ignited moxa-stick segment. The treatment in both groups lasted for 30 min every time, once every other day for 15 times, with 5 times being a therapeutic course and two days' rest between every two courses. The therapeutic effect was evaluated according to the "Criteria for Evaluating the Therapeutic Effect of Disorders of Traditional Chinese Medicine". The lumbago severity was assessed using visual analogue scale (VAS), and the lumbar functional activity assessed using modified Oswestry dysfunction (in objects-lifting, sitting, standing, walking, sleeping, pain, activity of daily living, social activity, outing, etc.) index (ODI). The plasma ß-EP content was assayed using ELISA. RESULTS: After the treatment, of the two 30 cases in the conventional acupuncture and warming needle moxibustion groups, 8 (26.67%) and 12 (40.00%) were under control, 8 (26.67%) and 10 (33.33%) had a marked improvement, 3 (10.00%) and 4 (13.33%) were effective, and 11 (36.66%) and 4 (13.33%) ineffective, with the effective rates being 63.34% and 86.66%, respectively. The effective rate of the warming needle moxibustion was significantly higher than that of the conventional acupuncture (P<0.05). The VAS scores and ODI values of the two groups were gradually decreased along with the prolongation of treatment, and significantly lower in the warming needle moxibustion group than in the acupuncture group at the 1st, 2nd and 3rd course of treatment (P<0.01). The levels of plasma ß-EP were gradually increased in the two groups at the 1st, 2nd and 3rd course of treatment, and were obviously higher in the warming needle moxibustion group than in the conventional acupuncture group at each of the 3 courses (P<0.01). No obvious adverse reactions were observed in the two groups. CONCLUSION: Warming needle moxibustion has a better therapeutic effect in relieving lumbago and lumbar dysfunction, and can up-regulate blood ß-EP level.