金线莲一促生真菌原生质体制备和再生研究

从药用植物内生真菌中筛选到对植物生长有显著促进作用的粘帚霉属的一种真菌（Ｇｌｉｏｃｌａｄｉｕｍｓｐ.简称Ｙ菌）,以它为出发菌株,进行原生质体制备与再生条件的研究。将培养４８ｈ的Ｙ菌菌丝体经过巯基乙醇处理３０ｍｉｎ,并用１％的纤维素酶和溶壁酶混合液于２８℃酶解３ｈ,原生质体得率可达２．１４Ｘ１０^７个／ｍＬ,在含０.５Ｍ的甘露醇为稳渗剂的再生培养基上,其原生质体的再生率可达３．８６ｘ１０^４。     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

绿木霉Gv29-8丝氨酸蛋白酶S8/S53超家族基因特性及功能

【背景】丝氨酸蛋白酶在木霉菌生物防治过程中发挥重要作用。【目的】研究绿木霉丝氨酸蛋白酶S8/S53超家族基因信息及其生物学功能,进而为该蛋白酶生防制剂的开发及基因改造提供理论支持。【方法】通过生物信息学分析方法,从绿木霉Gv29-8基因组中鉴定出23个丝氨酸蛋白酶基因,以少孢节丛孢菌ATCC 24927基因组中鉴定的4个丝氨酸蛋白酶基因作为对照,对这27个丝氨酸蛋白酶基因的特性、蛋白结构、进化地位、功能等进行预测分析。【结果】27个基因结构差异较大,编码的蛋白具有典型的丝氨酸蛋白酶催化三联体结构,属于S8/S53超家族,分为6个亚家族,同一亚家族的蛋白酶保守区长度相近,相似性较高,催化残基附近序列比较保守。系统进化分析显示,同一亚家族丝氨酸蛋白酶聚为一类。【结论】绿木霉和少孢节丛孢菌的部分丝氨酸蛋白酶基因在结构和蛋白性质上相似性强,亲缘关系较近,均属于S8_PCSK9_ProteinaseK_like亚家族,推测绿木霉与少孢节丛孢菌该亚家族的丝氨酸蛋白酶具有相似的功能,可抑制植物病原真菌和降解线虫体壁。     

Evaluating a diverse panel of biocontrol agents against infection of blueberry flowers by Monilinia vaccinii-corymbosi

Seven microorganisms were evaluated for their biocontrol potential against Monilinia vaccinii-corymbosi which causes mummy berry disease through gynoecial (stigma-style-ovary) infection of blueberry flowers: the bacteria Bacillus subtilis QRD137, B. mojavensis RRC101, B. mycoides 7IIC4, and Pantoea agglomerans C9-1S; the yeast Wickerhamiella australiensis Y-27360; and the filamentous fungi Trichoderma harzianum KRL-AG2 and Gliocladium roseum H47. The epiphytic fitness of each organism was investigated by evaluating population dynamics or fungal growth on the stigmas of detached blueberry flowers, and such flowers, co-inoculated with M. vaccinii-corymbosi, were used to determine efficacy in reducing pathogen infection of the style. In addition, all organisms were tested in vitro for antibiosis using dual cultures and for nutrient competition (niche overlap) using Biolog microplates. The most promising antagonists were P. agglomerans, which exhibited high epiphytic fitness on the stigma and consistently reduced stylar infection by the pathogen; B. subtilis, which showed strong antibiotic activity in vitro and considerably reduced pathogen ingress into styles, but whose limited epiphytic fitness decreases its potential for field-use; and G. roseum, which exhibited complete niche overlap with the pathogen in vitro but produced more variable results in reducing stylar infection. Future work should evaluate combinations of these antagonists to determine whether there are additive effects and whether the variability inherent in biocontrol can be reduced.     

Tidal,Daily, and Lunar‐Day Activity Cycles in the Marine Polychaete Nereis virens

The burrow emergence activity of the wild caught ragworm Nereis virens Sars associated with food prospecting was investigated under various photoperiodic (LD) and simulated tidal cycles (STC) using a laboratory based actograph. Just over half (57%) of the animals under LD with STC displayed significant tidal (～12.4 h) and/or lunar‐day (～24.8 h) activity patterns. Under constant light (LL) plus a STC, 25% of all animals were tidal, while one animal responded with a circadian (24.2 h) activity rhythm suggestive of cross‐modal entrainment where the environmental stimulus of one period entrains rhythmic behavior of a different period. All peaks of activity under a STC, apart from that of the individual cross‐modal entrainment case, coincided with the period of tank flooding. Under only LD without a STC, 49% of the animals showed nocturnal (～24 h) activity. When animals were maintained under free‐running LL conditions, 15% displayed significant rhythmicity with circatidal and circadian/circalunidian periodicities. Although activity cycles in N. virens at the population level are robust, at the individual level they are particularly labile, suggesting complex biological clock‐control with multiple clock output pathways.     

Chemical composition and characterization of a galactomannoglucan from Gliocladium viride wall material

The following fractions were obtained from the wall material of Gliocladium viride : F1 (27.5%), a glucan, containing xylose, mannose and galactose, coluble in 1 M NaOH at 20°C; F2 (6.7%), a β-glucan-chitin complex, solubilized with 1 M NaOH at 20°C from the previous residue left overnight at −20°C; F3 (8.1%), a glucan, containing mannose and galactose solubilized with 1 M NaOH at 70°C; and F4, the insoluble residue, a β-glucan-chitin complex similar to F2, amounting to 31.3% of the wall material.
F1 was extracted with distilled water. The soluble material (F1S) was a galactomannoglucan (54.7%) and the inscluble (F1P) a glucan (45.3%). Periodate oxidation revealed the presence of glycerol, erythritol, threitol, ribitol, arabitol, mannose, galactose and glucose in F1S, and glycerol and glucose as the main components in F1P. The fractions obtained when F1S was purified through Sepharose CL6B, were methylated.     

Biocontrol of Damping-off Diseases Caused by Rhizoctonia solani and Pythium ultimum with Alginate Prills of Gliocladium virens, Trichoderma hamatum and Various Food Bases

Alginate prills were formulated with the biomass of isolates of Gliocladium virens and Trichoderma spp. and various food bases (wheat bran, corn cobs, peanut hulls, soy fiber, castor pomace, cocoa hulls and chitin). Alginate prills with G. virens (Gl-21) biomass and all food bases except cocoa hull meal significantly reduced the damping-off of zinnia in a soil-less mix caused by Rhizoctonia solani and Pythium ultimum. The prills with bran, soy fiber, castor pomace or chitin resulted in stands similar to those in the non-infested control. In soil, prills with all the food bases and Thrichoderma hamatum (TRI-4) biomass controlled the damping-off of cotton caused by R. solani and gave stands comparable to, or better than, those in the non-infested control soil. Prills with all the food bases resulted in a proliferation of Gl-21 in a soil-less mix and of Gl-21 and TRI-4 in soil. Prills with food bases and TRI-4 biomass reduced the survival of R. solani in infested beet seed to less than 30%, with bran and chitin being the most effective food bases; prills with Gl-21 biomass and all food bases also reduced the survival of R. solani in beet seed, but not as much as did prills with TRI-4 biomass. In prills containing wheat bran, soy fiber or chitin, the biocontrol isolate Th-58 (T. harzianum) was almost as effective as TRI-4, but isolate Gl-3 (G. virens) was less effective. There was no significant interaction between the biocontrol fungus and the food base. The results suggest that the intrinsic properties of a selected fungus isolate are more important than some formulation variables in biocontrol.     

Extruded Granular Formulation with Biomass of Biocontrol Gliocladium virens and Trichoderma spp. to Reduce Damping-off of Eggplant Caused by Rhizoctonia solani and Saprophytic Growth of the Pathogen in Soil-less Mix

Extruded granular formulations containing rice flour, gluten, Pyrax, vermiculite, canola oil, and fermentor-produced biomass of isolates of Gliocladium virens (Gl-3, Gl-21 and Gl-32), Trichoderma hamatum (TRI-4 and 31-3), T. harzianum (Th-32 and Th-87) and T. viride (Tv-101) were evaluated for their effect on the reduction of eggplant damping-off caused by Rhizoctonia solani, reduction of pathogen inoculum and proliferation of the isolates in a soil-less mix. Granules with all isolates except 31-3 significantly (P < 0.01) reduced damping-off, and granules with Gl-3, Gl-21, Gl-32, TRI-4 and Th-87 yielded stands comparable to that (90%) of the non-infested control. Granules with isolates Gl-21 and TRI-4 were the most effective in the reduction of saprophytic growth of R. solani, and there was a significant inverse correlation (r ² = - 0.82) between eggplant stand and saprophytic growth of the pathogen over all treatments. Isolate propagules proliferated to about 10⁷ colony-forming units (CFU) g^?1 of soil-less mix after a 6-week incubation, but there was no correlation between the number of CFU and eggplant stand or saprophytic growth reduction of the pathogen. Granules with Gl-21 and TRI-4 amended to pathogen-infested soil-less mix at a rate as low as 0.06% significantly (P < 0.05) reduced damping-off and pathogen saprophytic growth, and a rate of 0.25% of Gl-21 granules resulted in an eggplant stand comparable to that of the non-infested control. There was no significant correlation between the rate of granule amendment and the proliferation of Gl-21 and TRI-4. Granules of Gl-21 and TRI-4 also significantly prevented the spread of R. solani in flats of eggplant seedlings when the biocontrol granules were applied to the soil-less mix 1 day before the pathogen inoculum.     

Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in Non-sterile Potting Medium

Interactions of Gliocladium virens with Pythium ultimum and Rhizoctonia solani under simulated in vivo conditions were observed microscopically. Different types of propagules of the three fungi were paired on nitrocellulose membranes and incubated at 25°C in non-sterile potting medium in Petri dishes for 1-5 days. Alginate-wheat bran prill were used as carriers for G. virens. Prill inoculated with G. virens and pre-incubated in potting medium for 3-5 days before placement on membranes did not inhibit the germination of Pythium sporangia, but subsequent Pythium growth was markedly stunted and distorted, with some hyphal collapse and cytoplasmic leakage. G. virens had no visible effect on older Pythium mycelium. Two to 5 days' growth of G. virens caused cytoplasmic leakage of Rhizoctonia mycelium, prevented secondary branching of hyphae and occasionally coiled around Rhizoctonia hyphae. Prill that were newly colonized by G. virens, but not prill pre-incubated for 3 or 5 days, stimulated the growth of Pythium mycelium and sporangia, Rhizoctonia mycelium and unprimed monilioid cells, probably by supplying nutrients. The timing of the interactions and their specificity for the different pathogen propagules were consistent with the production of gliotoxin by G. virens. This view was supported by in vitro experiments, in which pathogen propagules were incubated in a range of concentrations of gliotoxin in potato dextrose broth. Pythium sporangia and mycelium were inhibited by 1 or 2 μmg ml^-1, but Rhizoctonia monilioid cells and mycelium required 3-5 μmg ml^-1 for inhibition. At the lowest effective concentrations the inhibition was sometimes reversible, but propagules were killed at high concentrations of gliotoxin.