Effect of Inoculum Rates and Sources of Coniothyrium minitans on Control of Sclerotinia sclerotiorum Disease in Glasshouse Lettuce

Coniothyrium minitans isolate Conio grew on both maizemeal-perlite and ground maizemeal-perlite, producing high numbers (1.6×10⁷ conidiag^–1 inoculum) of germinable conidia. Coniothyrium minitans isolate Conio applied as a preplanting soil incorporation of maizemeal-perlite inoculum at full application rate (0.6lm^–2; 10¹¹ colony forming units (cfu)m^–2) significantly reduced Sclerotinia disease in a sequence of three lettuce crops grown in a glasshouse. No reduction in disease was achieved with any of the reduced rate treatments (10⁸cfum^–2) of a range of C. minitans isolates (Conio ground maizemeal-perlite at reduced rate, Conio and IVT1 spore suspensions derived from maizemeal-perlite, IVT1 spore suspension derived from oats and Contans® WG spore suspension). After harvest of the second and third crops, C. minitans maizemeal-perlite at full rate reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore suspension and reduced rate maizemeal-perlite inocula. Coniothyrium minitans was recovered from the soil throughout the trial, between 10⁵ and 10⁷cfucm^–3 in maizemeal-perlite inoculum full rate treated plots and 10¹–10⁴cfu cm^–3 in all other inoculum treated plots.Pot bioassays were set up corresponding to the inoculum used in the glasshouse, with the addition of Conio ground maizemeal-perlite at a rate corresponding to the full rate maizemeal-perlite. Coniothyrium minitans maizemeal-perlite and ground maizemeal-perlite at full rate significantly decreased carpogenic germination, recovery and viability of sclerotia and increased infection of sclerotia by C. minitans in comparison with spore suspension treatments, reflecting results of the glasshouse trials. Additionally, reduced maizemeal-perlite treatment also decreased apothecial production, recovery and viability of sclerotia compared with the spore suspension treatment, despite being applied at similar rates. Simultaneous infection of sclerotia by several isolates of C. minitans was demonstrated. Inoculum level in terms of colony forming unitscm^–3 of soil appears to be a key factor in both control of Sclerotinia disease and in reducing apothecial production by sclerotia.     

枯草芽孢杆菌G3菌株抑制立枯丝核菌菌核形成的影响因子分析

产几丁质酶枯草芽孢杆菌G3菌剂在土壤中可抑制水稻纹枯病菌菌核形成。菌剂的适宜剂量为 1 1 5× 1 0 8cfu/g。温度、土壤含水量和菌剂处理的时间是影响效果的因素。 1 5～ 30℃培养 ,G3菌剂处理的菌核减退率从 35 2 %逐步上升到 77 8%。在 1 6 %～ 2 4 %土壤含水量的条件下 ,菌核减退率从 75 5%下降到 2 1 7%。接种病原真菌前施用G3菌剂比接种后效果更好。来源于湖南、山西、云南和上海的不同区域土壤对防效没有显著影响。菌剂添加豆粕粉、壳聚糖、葡萄糖和淀粉有不同程度的增效作用 ,尤其是壳聚糖 ,增效率达 2 9 2 %。     

Biotrophic mycoparasitism byVerticillium biguttatum onRhizoctonia solani

Verticillium biguttatum cannot utilise cellulose or nitrate-nitrogen and it requires biotin for growth, yet it grew and sporulated abundantly onRhizoctonia solani on cellulose, obtaining at least organic carbon, nitrogen and biotin fromR. solani. Videomicroscopy of inter-hyphal interactions on films of water agar showed thatV. biguttatum behaved as a biotrophic mycoparasite. From germinating spores, it penetrated the hyphae ofR. solani and formed haustorium-like branches without killing the host cells, and the haustoria supported an external mycelial network of the mycoparasite. Later the mycoparasite sporulated, and the infected host cells died. On cellulosic substrataV. biguttatum did not reduce the growth ofR. solani, and often enhanced the rate of cellulose degradation. However,V. biguttatum drastically reduced the production of sclerotia byR. solani, often completely suppressing sclerotium production when the mycoparasite infected only a localized region of the host colony. This is ascribed to the creation of a nutrient sink by the parasite, consistent with biotrophy. On plates of cellulose agar the suppression of sclerotia was not confined to parasitized colonies but extended to adjacent colonies ofR. solani that had successfully anastomosed with the parasitized colony. There was no effect on adjacent vegetatively incompatible colonies, where attempted anastomoses caused cytoplasmic death. In comparable experiments the necrotrophic mycoparasiteGliocladium roseum had no long-distance effect on sclerotium production byR. solani.Suppression of sclerotium production may explain the reported success ofV. biguttatum in biocontrol of black scurf of potato in experimental field conditions.     

盾壳霉对核盘菌的拮抗作用研究

系统研究了菌核重寄生菌盾壳霉对核盘菌的拮抗作用,结果表明:盾壳霉可在核盘菌菌落上寄生,使核盘菌菌丝消解、原生质泄露,并抑制菌核的形成;而且盾壳霉提前6d接种,其分泌的抗生物质还可抑制核盘菌菌丝生长,并产生明显抑菌带。盾壳霉孢子液喷雾处理也证明:盾壳霉分生孢子即可在核盘菌子囊盘(柄)上萌发、寄生,使子囊盘(柄)萎缩枯死,而且还可以在核盘菌菌落上萌发、寄生,消解破坏菌丝体、抑制的菌核形成。     

The effect of temperature and water potential on the production of conidia by sclerotia of Botrytis squamosa

The rate of conidiogenic germination of Botrytis squamosa was highest at 16°C and the greatest numbers of conidia per sclerotium (up to 5 × 10⁴) were produced at temperatures of 5–10°C. At temperatures above 20°C, the percentage of sclerotia producing conidia declined rapidly. Decreasing water potential reduced the rate at which conidia were produced and also resulted in fewer conidia produced per sclerotium. However, conidia were produced at water potentials as low as −2 MPa, at which sclerotial germination was at least 60%. A simulation model that included effects of both temperature and water potential was developed from laboratory and field data obtained for conidial production in sclerotia exposed for periods of 1, 2, 3 or 4 weeks during an entire year. There was good agreement between conidiogenic germination predicted by the model and conidial production observed in onion plots artificially inoculated with sclerotia. Temperature and water potential were therefore considered to be the principal microclimatic factors affecting conidial production by B. squamosa. The role of sclerotia in the context of UK onion production is discussed.     

翻面法培养白绢病菌菌核的研究

采用翻面法对白喙病菌最适培养基的最、最佳翻面的时机等进行了研究,结果表明：翻面法的最适培养基使用量,以每个直径为８５ｍｍ培养皿使用２０ｍｌ为佳,培养的菌核数量多,成熟度一致;翻面时机为菌丝长满平板时最佳,形成的菌核数量多,成熟快。     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: control of sclerotinia disease in glasshouse lettuce

The effects of Coniothyrium minitans inoculum quality and an 8-week interval between inoculum application and crop planting on sclerotinia ( Sclerotinia sclerotiorum ) disease in three successive lettuce crops were investigated in a glasshouse trial. Spore suspensions of three isolates of C. minitans (Conio, IVT1 and Contans) applied at 10⁸ CFU m⁻² and a standard Conio maizemeal–perlite application (06 L m⁻², 10¹¹ CFU m⁻²) were assessed for their ability to control S. sclerotiorum . Only the maizemeal–perlite inoculum (isolate Conio) consistently reduced sclerotinia disease. In the third lettuce crop only, isolates IVT1 and Contans formulated by Prophyta and isolate IVT as an oil–water formulation, all applied as spore suspensions, reduced disease at harvest compared with the untreated control. Recovery, viability and C. minitans infection of sclerotia buried during the 8-week period prior to each of the three lettuce crops, and of sclerotia formed on the crop, were tested. Only the maizemeal–perlite inoculum (isolate Conio) reduced the recovery of sclerotia buried in soil for weeks between inoculum application and crop planting, reducing their viability and increasing infection by C. minitans . Eight weeks was sufficient to enable C. minitans to infect sclerotia of S. sclerotiorum , and may account for disease control. After harvest of the second and third crops, maizemeal–perlite treatment (isolate Conio) reduced the number and viability of sclerotia recovered on the soil surface and increased infection by C. minitans compared with spore-suspension treatments. The effect of inoculum concentration and the influence of soil temperature (varying with time of year) on infection of sclerotia by C. minitans are discussed.     

生菜软腐和菌核病拮抗菌贝莱斯芽胞杆菌BPC6鉴定与防效

软腐病和菌核病是生菜生产中两大毁灭性病害。为有效控制这2种病害，利用平板稀释和对峙法，从生菜根际土壤中分离到一株拮抗菌BPC6，其菌悬液、无菌滤液对软腐病菌Pectobacterium carotovorum和菌核病菌Sclerotinia sclerotiorum的生长均具有抑制效果，该菌株也能抑制其他14种植物病原菌的生长。形态学、16S rDNA及基因组序列分析将其鉴定为贝莱斯芽胞杆菌Bacillus velezensis。BPC6能产生纤维素酶和蛋白酶，不能产生几丁质酶和嗜铁素。比浊法分析的生长曲线显示，BPC6能在1%～10% NaCl及pH 5.0～9.0条件下生长。通过分析不同浓度BPC6对软腐病菌和菌核病菌侵染生菜离体叶片的影响，明确了菌株BPC6适宜用量为1.4×10⁹～2.8×10⁹ CFU/mL。菌株BPC6对生菜软腐病和菌核病盆栽防效分别为44.09%和53.58%，对菌核病大田防效为77.41%。本研究表明BPC6是一株对生菜软腐病和菌核病具有拮抗作用的潜在生防菌。     

Method for Estimating the Content of the Chloroform-Extractable Green Fraction (CEGF) in HCl-DMSO Extract of Soils

Chloroform-extractable green fraction (CEGF) was detected in the supernatant obtained by alkali precipitation from the HCI-dimethylsulfoxide (DMSO) extract of Pg-rich soil. In the alkaline solution, the color of CEGF was green and CEGF showed strong Pg-like absorption bands. Ultraviolet and visible (UV-VIS) spectral analysis and gel chromatography on Sephadex G-50 were performed to compare several properties between CEGF and Pg. CEGF, which was purified by gel chromatography on Bio-Beads SX-1, displayed strong absorption bands at 609, 562, 445 and 280 inn in the alkaline solution. These absorption bands were almost similar to those of Pg. Furthermore, the UV-VIS spectrum of CEGF in the organic solvents showed a similar characteristic pattern of 4,9-dihydroxyperylene-3,10-quinone (DHPQ), which was considered to be a chromophore of Pg. Based on the results of gel chromatography on Sephadex G-50, CEGF mainly consisted of two fractions, corresponding to the G₂ and G₃ fractions of Pg. These results indicated that the method for extracting CEGF from Pg-rich soil in the present study was easy and selective and that CEGF was one of the components of, or a closely related substance to Pg. A colorimetric method for the estimation of the CEGF content in soils was developed. The calibration curve of CEGF was linear over a wide range of contents from 2.75 to 220 mg L⁻¹. The CEGF content in twelve samples of various soils was examined. CEGF was detected in all the soil sampled (5 orders) including three samples (3 orders) where Pg was not detected, and the content ranged from 0.07 to 1.66 g kg⁻¹ (dry soil). Therefore, the method for estimating the CEGF content in soils developed in the present study was found to be suitable for various soil orders and it was assumed that CEGF occurred in various soil orders.     

Aluminum concentrations in sclerotia from a buried humic horizon of volcanic ash soils in Mt. Myoko,Central Japan

The morphology and chemical composition of fungal sclerotium-like grains collected from the buried humic horizon of volcanic ash soil in Mt. Myoko were examined using a scanning electron microscope (SEM), energy dispersion xray micro-analyzer (EDX), and electron probe micro-analyzer (EPMA). A high C content, low level of Si and high concentration of Al characterized the grains considered to correspond to the sclerotia of Cenoccocum graniforme. The ²⁷Al MAS NMR spectrum of the sclerotia showed the existence of 6- and 4-coordinated Al. X-ray diffraction analyses supported the presence of amorphous Al (ex. Al-humus complex) in the sclerotia.