Mechanisms of Trichoderma longibrachiatum T6 Fermentation against Valsa mali through Inhibiting Its Growth and Reproduction, Pathogenicity and Gene Expression.

Apple Valsa canker is one of the most serious diseases, having caused significant apple yield and economic loss in China. However, there is still no effective biological methods for controlling this disease. Our present study focused on the inhibitory activity and mechanisms of Trichoderma longibrachiatum (T6) fermentation on Valsa mali that causes apple Valsa canker (AVC). Our results showed that the T6 fermentation exhibited effective antifungal activity on the mycelial growth and conidia germination of V. mali, causing mycelium malformation and the hyphal disintegrating in comparison to the control. The activity of pathogenically related enzymes that are secreted from V. mali and the expression level of gene of V. mali were significantly inhibited and downregulated by treatment with T6 fermentation. In addition, the lesion area and number of pycnidia of V. mali formed on the branches were significantly reduced after treatment with the T6 fermentation through the pathogenicity test on the detached branches. Our results indicate that the possible mechanism of T6 fermentation against V. mali occurs through inhibiting its growth and reproduction, the pathogenic enzyme activity, and its related gene expression.

Mycobiota of maize seeds revealed by rDNA-ITS sequence analysis of samples with varying storage times.

Fungi are an integral component of the plant microbiome. However, the composition and variation in the fungal communities (mycobiota) associated with seeds are poorly understood. In this study, we investigated the mycobiota of 11 maize seed samples with storage times ranging from 6 months to 12 years. Mycobiota were characterized by a culture-based approach, and fungal species were identified through rDNA-ITS sequence analyses. From a total of 169 pure fungal isolates obtained from both the seed surface and internal tissues, we identified 16 distinct species (belonging to 10 genera) associated with maize seeds, all but one of which were ascomycetes. Among these species, seven were exclusively isolated from internal tissues, two species were isolated only from the seed surface, and another six species were isolated from both the surface and internal tissues. Aspergillus niger was consistently found under all storage conditions and dominated fungal communities with a relative abundance of 36%-100%. Species of Fusarium (9%-40%) and Penicillium (9%-20%) were also frequently isolated, but other species appeared sporadically and were isolated from fewer than three seed stocks. According to our results, while the overall incidence of fungal infection generally declined with storage time, there was no consistent association between seed storage time and fungal species richness or relative abundance; furthermore, the composition of the mycobiota associated with maize seeds was highly variable among the samples. The detection of the four major mycotoxigenic fungal genera, specifically Aspergillus, Fusarium, Penicillium, and Alternaria, was alarming, and the isolation of a potential controlling agent as well as information about their temporal occurrence will contribute to the management of mycotoxins in the future.

[Efficiency of Trichoderma longibrachiatum T6 in the control of Meloidogyne incognita and its rhizosphere colonization in cucumber].

Efficiency of different concentrations of Trichoderma longibrachiatum T6 against Meloidogyne incognita and its rhizosphere colonization in cucumber were determined in greenhouse experiments. The results of rhizosphere colonization experiments showed that the number of colonies in cucumber soil and root increased significantly ten weeks after inoculation with the second stage juveniles of M. incognita and different concentrations of T. longibrachiatum T6, and there was significant difference in different concentrations of T. longibrachiatum T6, e.g., the maximum numbers of colonies in soil and root were 7.8 x 107 and 6.3 x 105 CFU · mL⁻¹ respectively after treated with the spore suspension of 1.5 x 107 CFU · mL⁻¹. Greenhouse experiments results showed that different concentrations of T. longibrachiatum T6 had significant control effect on different life stages of M. incognita, and the control effect increased with the concentration of T. longibrachiatum T6. T. longibrachiatum T6 significantly increased plant height, root length, above-ground and root fresh mass o cucumber inoculated by M. incognita. T. longibrachiatum T6 could colonize in cucumber rhizosphere, had control effect on M. incognita, and significantly improved the growth of cucumber.

[Screening, identification and biocontrol effect of antagonistic actinomycetes against the pathogen of Cytospora sp. for apple tree]. / è‹¹æžœæ ‘è çƒ‚ç— èŒæ‹®æŠ—æ”¾çº¿èŒçš„ç­›é€‰ã€é‰´å®šåŠé˜²æ•ˆ.

The pathogen of Cytospora sp. was considered as the target fungus to evaluate the bio-control potential of antagonistic actinomycetes against Cytospora sp. in the present study, which was isolated from the rhizosphere soil of apple tree. Moreover, the antagonistic effect of actinomycetes against Cytospora sp. growth was determined by the method of confrontation and growth rate, and the screened antagonistic strain was identified by the method of morphology and molecular biology. Also, the inhibitory activity of strain ZZ-9 against the conidium germination and mycelia growth of Cytospora sp. was determined in vivo, and its bio-control effect on Cytospora sp. growth was determined in detached apple twigs. The results showed that fifteen strains of actinomycetes had the inhibitory effect on Cytospora sp. growth, among all the isolated strains, which accounted for 18.8% and especially the inhibitory rate of eight strains was more than 50%. In addition, among all the screened strains, the strain ZZ-9 presented the highest inhibitory rate of 96.4%, which was significantly higher than those of the other isolated strains. The strain ZZ-9 was initially identified as Streptomyces rochei according to cultural characteristics, physiological and biochemical properties and 16S rDNA analysis. The nucleotide sequences in GenBank accession number was registered as KT986228. Different dilution of ZZ-9 fermentation had significant inhibitory effect on Cytospora sp. conidium germination and mycelia growth. The inhibitory rates of the 50 times fermentation on both conidium germination and mycelia growth were more than 80%. The inhibited mycelia's color was deepened, the mycelia branches were increased, and the ends of hyphae were swelled and deformed, even the protoplasm was concentrated and released. The bio-control effect of the ZZ-9 stock solution on Cytospora sp. growth was more than 75% in detached apple twigs. Thus, our results indicated that the strain ZZ-9 could be used for controlling apple tree Valsa canker in vivo and vitro.

[Effects of Yishen Shengjing Capsules on semen quality and gonadal hormone levels in rats with dibutyl phthalate-induced reproductive function injury].

OBJECTIVE: To study the possible pathogenesis of infertility caused by dibutyl phthalate (DBP) and investigate the effects of Yishen Shengjing Capsules (YSC, kidney-tonifying and essence-producing capsules) on DBP-induced reproductive function injury and its possible action mechanisms in male Wistar rats. METHODS: Models of DBP-induced reproductive function injury were made in 80 male Wistar rats and another 20 were used as blank controls. After modeling, the model rats were randomly divided into a model control, a high-dose YSC, a medium-dose YSC, and a low-dose YSC group. Four weeks after intervention, all the animals were sacrified for observation of the histomorphologic changes in the testis under the light microscope, measurement of sperm concentration, motility and abnormality, and determination of the levels of serum testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by radioimmunoassay. RESULTS: Compared with the blank controls, the model rats showed obvious pathological changes in testicular histomorphology, significantly decreased sperm concentration and motility, increased sperm abnormality, reduced contents of serum T and LH, and elevated the level of serum FSH (P<0.01). After 4 weeks of medication, the animals of the high-, medium-, and low-dose YSC groups, in comparison with the model controls, exhibited different degrees of recovery from testicular histomorphological damage, remarkably increased sperm concentration and motility, decreased sperm abnormality, elevated levels of serum T and LH, and reduced content of serum FSH (P<0.01). There were statistically significant differences in all the parameters above between the high-dose YSC and medium- and low-dose YSC groups (P<0.01). CONCLUSIONS: DBP reduces sperm motility and concentration, increases sperm abnormality, causes damage to the morphological structure of the rat testis, decreases the contents of serum T and LH, and elevates the level of the serum FSH. Yishen Shengjing Capsules can improve DBP-induced productive function injury, increase sperm motility and concentration, decrease sperm abnormality, elevate the level of serum T and LH, reduce the content of serum FSH, improve the morphological structure of the testis, and thus promote the reproductive function of the male rat.

[Lethal effects of Trichoderma longibrachiatum on Heterodera avenae].

The lethal effect of the conidia suspension of Trichoderma longibrachiatum against second stage juveniles of Heterodera avenae was observed under microscopic conditions and studied in vitro to preliminarily determine the potential and mechanism of the conidia suspension of T. longibrachiatum against H. avenae. Microscopic observation results showed that the conidia suspension of T. longibrachiatum adhered to or parasitized on the surface of second stage juveniles, even some parasitized nematodes were deformed at the initial stage. Later, the second stage juveniles were wrapped and the integuments were penetrated by a large number of hyphae germinated from the conidia suspension of T. longibrachiatum. Even the body of dead second stage juveniles was completely lysed. In vitro studies showed that different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum (conidia suspension) had strong lethal and parasitic effects on the second stage juveniles of H. avenae, and significant differences existed among the treatments with different concentrations of T. longibrachiatum. In addition, the lethal and parasitic effects increased with increasing the T. longibrachiatum concentration. The mortality and corrected mortality of the second stage juveniles treated with the concentrations of 1.5 x 10(7) cfu x mL(-1) were 91.3% and 90.4% after 72 hours, and the second stage juveniles were parasitized by 88.7% after 14 days. Therefore, the conidia suspension of T. longibrachiatum had a great lethal effect on H. avenae, and the strain could be considered as a potential bio-control agent against H. avenae.

[Parasitic and lethal effects of Trichoderma longibrachiatum on Heterodera avenae: microscopic observation and bioassay].

A laboratory experiment was conducted to study the parasitic and lethal effects of Trichoderma longibrachiatum conidia suspension on Heterodera avenae cysts. Different concentrations (1.5 x 10(5)-1.5 x 10(7) cfu x mL(-1)) of T. longibrachiatum conidia suspension had strong parasitic and lethal effects on H. avenae cysts, and the effects differed significantly among the different concentrations. When treated with the T. longibrachiatum conidia suspension at a concentration of 1.5 x 10(7) cfu x mL(-1), 96.7% of the H. avenae cysts were parasitized by the conidia at the 18th day, and the hatching rate of the cysts was inhibited by 91.2% at the 22nd day. The microscopic observation showed that at the initial parasitic stage, T. longibrachiatum conidia suspension adhered or parasitized on the cyst surface, germinated a large number of hyphae, and grew on the cyst surface, making the development of cyst embryo stopped and the contents in cysts flocculated, and even, some cysts started to deform, and small dark brown vacuoles formed on the cyst surface. At the later parasitic stage, the cysts were penetrated by dense mycelium, cysts were broken, their contents exosmosed, and the mycelium on the integument of some cysts produced conidiophores, on which, conidium were adhered or parasitized. It was considered that T. longibrachiatum could be used as a potential high-efficient bioagent to control the occurrence and damage of H. avenae.

[Effects of Qiangjing Capsule on the oxidative and antioxidative system in the epididymis of varicocele rats].

OBJECTIVE: To observe the effects of Qiangjing Capsule (QC) on the oxidative amd antioxidative system in the epididymis of varicocele rats in comparison with those of Shaofuzhuyu Capsule (SC) and Wuziyanzong Capsule (WC), and to explore its possible mechanism of enhancing epididymal sperm maturation. METHODS: Ten of 100 adolescent male SD rats were randomized to a sham-operation group, and varicocele models were successfully established in 72 of the other 90 by narrowing of the left renal vein. Then the model rats were equally assigned to 6 groups: model control, high-dose QC (0.216 g/ml), medium-dose QC (0.108 g/ml), low-dose QC (0.054 g/ml), SC (0.146 g/ml), and WC (0.130 g/ml). After 4 weeks of treatment, we determined the activity of glutathione peroxidase (GPx) and the level of malondialdehyde (MDA) in the left epididymis of different groups of rats. RESULTS: Compared with the sham-operation group, the model group showed a significant decrease in GPx activity (P < 0.01) and a marked increase in the MDA level (P < 0.05). And the high-dose QC group exhibited a significantly hither GPx activity and lower MDA level than all the other groups (P < 0.01 and P < 0.05). CONCLUSION: Varicocele can reduce the activity of GPx and elevate the level of MDA in the epididymis of rats, while Qiangjing Capsule can increase the former and decrease the latter, and thereby may improve epididymal microenvironment, enhance epididymal sperm maturation and promote fertility.

[Xiaojin Wan inhibits the expression of COX-2 in prostate tissues of prostatitis pain rats].

OBJECTIVE: To study the effect of Xiaojin Wan on the expression of COX-2 in the prostate tissues of rats with prostatitis pain, and the action mechanism of the drug alleviating the prostatitis pain. METHODS: Sixty male Wistar rats were randomized into two groups, 10 as blank controls, injected with aqua pro injection into the ventral part of prostate, and the other 50 as prostatitis pain models, given complete Freund's adjuvant (CFA). Three days later, the pain model rats were again equally divided into 5 subgroups: model control, Celecoxib Capsules, high-, median- and low-dose Xiaojin Wan, receiving intragastric administration of distilled water, Celecoxib Capsules and different doses of Xiaojin Wan respectively for 4 weeks. Then they were killed, the harvested tissues fixed with 10% paraformaldehyde and the changes of the COX-2 expression in the prostate detected with the immunohistochemical technique and graphics video analysis system. RESULTS: The expression of COX-2 was strong in the model group, significantly lower in the high- and median-dose and the Celecoxib Capsules groups than in the model control (P < 0.01) as well as in the high-dose than in the median- and low-dose groups (P < 0.01). CONCLUSION: Xiaojin Wan may alleviate prostatitis pain by inhibiting the expression of COX-2 in prostate tissues.

[Angiogenesis and regulatory factors in rats with BPH induced by testosterone].

OBJECTIVE: To study angiogenesis and regulatory factors in the proliferated prostatic tissues of Sprague Dawley (SD) rats with BPH induced by testosterone. METHODS: Sixteen castrated SD rats, aged 8 weeks and weighing 200 approximately 250 g, were equally randomized into a model group and a control group, and the BPH model was established by subcutaneous injection of testosterone. Immunohistochemistry and MIAS (micro-image analysis system) were used to test the manifestations of MVD (microvessel density), VEGF (vascular endothelium growth factor), flk-1, endostatin, MMP-2 (matrix metalloproteinase-2) and TIMP-2 (tissue inhibitor of metalloproteinase-2) in the prostatic tissues of both the model and the control groups. Multiple linear regression with the stepwise method was adopted to analyze the data. RESULTS: The manifestations of MVD, VEGF, flk-1, MMP-2, MMP-2/TIMP-2 and VEGF/endostatin in the model group were higher, while that of endostatin was lower than in the control group (P < 0.01), and the manifestation of TIMP-2 showed no statistical difference between the two groups. The regression analysis indicated that MVD was positively correlated to VEGF, VEGF/endostatin and MMP-2/TIMP-2 (r = 0.974, 0.986, 0.982, P < 0.05) and negatively correlated to endostatin (r = - 0.975, P < 0.05) . CONCLUSION: Testosterone could induce BPH in SD rats by increasing MVD and promoting the multiplication of vascular endothelial cells after regradation of basement membrane.

[The microstructure studies of penile cavernous tissue of erectile dysfunction rat models with chronic renal failure].

OBJECTIVE: To investigate the mechanism of erectile dysfunction (ED) in rat models with chronic renal failure (CRF). METHODS: Chronic renal failure was induced by adult male Sprague-Dawley rats, which were subjected to an excisional 5/6 nephrectomy. The rats in NCRF group and CRF group were divided into three groups randomly. Injected with apomorphine(APO, 80 microg/kg), penile erections of three groups were observed and noted by the 2nd week, 4th week respectively and 6th week from the 14th day of 5/6 nephrectomy and experimental models of ED with CRF were selected; NOS activity was examined and the microstructures of penile were observed under optical microscope with computer configuration image analysis system in selected rat models. RESULTS: Compared with the controls, the areas of smooth muscle and NOS activity in the penile cavernous tissue of ED rats with CRF decreased significantly (P < 0.01 or P < 0.05) , and collagen fibers slightly increased, and these alterations had close correlations with the duration of CRF. The CONCLUSIONS: Penile erection is seriously affected in rats with CRF. The decreases of areas of blood sinus had no obvious changes. the number of smooth muscles and NOS activity might be the most important factors.

[Experimental and clinical research of "Keyouling" on treatment of condyloma acuminata].

OBJECTIVE: To investigate the effects of traditional Chinese medicine "Keyouling" in treatment of condyloma acuminata (CA). METHODS: The effects of "Keyouling" at different concentrations on growth and proliferation of rabbits' prepuce epithelial cells and CA warts tissue were assessed by means of cellular primary culture and subculture, and the effects of "Keyouling" at different concentrations on the expression level of HPV6.11 DNA and expression level of HPV6.11 DNA of different area of CA patient's penis were quantitatively detected by means of FQ-PCR. RESULTS: "Keyouling" had no effect on growth and proliferation of rabbits' prepuce cuticle cells, but it had significant inhibition on CA warts tissue cells and expression of HPV6.11 DNA. Concentrations of "Keyouling" assumed negative correlated with cell proliferation rate of CA warts tissue and expression level of HPV6.11 DNA, and also had significant inhibition on expression of HPV6.11 DNA of negative cutaneous lesion area and cutaneous lesion area of CA patients. CONCLUSION: "Keyouling" had significant anti HPV6.11 effect, could significantly restrain growth and proliferation of CA warts tissue cells, while it had no damaging effect on normal organic cuticle cells. It was suggested that "Keyouling" was safe in clinical use and had good therapeutic effect on CA and subclinical infection.

[Therapeutic mechanisms of traditional Chinese medicine for chronic prostatitis].

Traditional Chinese medicine (TCM) has its unique therapeutic efforts on chronic prostatitis, a common disease of the adult male. This article systematically reviews several aspects below: anti-inflammation, anti-infection, anti-fibrosis, micro-circulation improvement, urodynamics improvement, immunomodulation, improvement of the pH value of prostatic fluid and percolation dynamics of prostatic drugs, adjustment of the microelement zinc, and the characteristics of TCM therapy. It also points out the gaps in recent researches and the focal points of future development.

[New progress in etiological factor study and treatment of erectile dysfunction in chronic renal failure patients].

Patients with chronic renal failure (CRF) experience a significant decrease in their living quality, due to the limitations imposed by the disease as well as the demands of the treatment that they receive. Some side effects contribute to the morbidity of these patients. Among them, erectile dysfunction (ED) is notable. This article reviews recent advances in etiological factor study and treatment of ED in CRF patients, which may guide the clinic practices in a considerable scale.

[Clinical application research of feilinjian peroral liquid in the treatment of Chlamydia and Mycoplasma infectious prostatitis].

OBJECTIVES: To evaluate the validity and security of Feilinjian peroral liquid in the treatment of chlamydia trachomatis (CT), mycoplasma hominis (Mh) and Ureaplasma urealyticum (UU) infectious prostatitis. METHODS: Two hundred and three patients of CT, Mh or UU infectious prostatitis diagnosed by strict orientation examination were randomly divided into two groups, one given feilinjian peroral liquid, and the other azithromycin perorally, for one month as treatment period. RESULTS: The analytical results showed that 54 (42.86%) were clinically fully recovered, 32(32.39%) were significantly effective, and 13 (10.31%) were effective in feilinjian peroral liquid group, with total significantly effective rate 68.25% and total effective rate 78.57%, both obviously higher than those in the control group(50.64% and 57.14% respectively). CONCLUSIONS: Feilinjian peroral liquid is significantly better than azithromycin in ameliorating self-conscious symptoms, symptoms scoring of patients with CT, Mh and UU infection, with little side-effects.

[Histomorphological studies on hyperplastic prostate of castrated rat caused by androgen].

OBJECTIVES: To investigate prostatic histomorphological changes of rat benign prostatic hyperplasia (BPH) models. METHODS: After castrated, SD rats were injected subcutaneously testosterone propionate to induce the BPH. Water substitution method was used to measure prostatic volumes. Prostatic tissue were stained by hemoloxylin and eosin, the morphometric changes of glandular and interstitial tissues were semi-quantified by image analysis system. Multiple linear regression was adopted to analyze the results. RESULTS: In comparison with normal group, prostatic volumes were significantly enlarged (P < 0.01) with glands expanded and interstitial tissues increased in BPH model. Glandular average diameters, volumes and surface areas in unit volume, as well as glandular circumferences and glandular relative total volumes were all significantly increased (P < 0.01). Glandular counts, density, ratio of glandular surface area to volume. And glandular average curvature were all declined (P < 0.05-0.01), so was interstitial circumference (P < 0.01). But volume density had no changes, and relative total interstitial volumes were obviously increased. Prostatic volume was significantly correlated with glandular relative total volume (r = 0.989, P < 0.001) and interstitial relative total volume(r = 0.789, P < 0.001). Prostatic volume was also correlated significantly with glandular average volume(r = 0.789, P < 0.001). CONCLUSIONS: Action of androgen on rat prostate may mainly lie in glandular epithelial hyperplasia, which manifests enlargement of glandular lumen accompanied by hyperplasia of interstitial tissues.