Mycelium of Alternaria alternata as a potential biological control agent for Eupatorium adenophorum

The fungus, Alternaria alternata (Fr.) Keissler Strain 501, has been evaluated as a bioherbicide for control of Eupatorium adenophorum Spreng., but the biology of the pathogen–host interaction and the optimal environmental conditions for disease development and effective weed control are unknown. Disease development of A. alternata Strain 501 mycelia on E. adenophorum was assessed under several factors including pathogen inoculum concentration, plant age, dew period duration, post-dew temperature, storage temperature and duration. The minimum inoculum concentration required to kill E. adenophorum seedlings was 3.2×10⁶ mycelial fragment mL^?1. E. adenophorum seedlings at the four-leaf-pair stage were more susceptible than the older plants, especially those at the older than seven-leaf-pair stage. With a dew period of at least 14 h, 100% mortality occurred. The optimal post-dew temperature for disease development was 18–25°C. Storage at <4°C maintained the infectivity of A. alternata strain 501 mycelia on E. adenophorum longer. Using light and scanning electron microscopy to examine the infection process of A. alternata Strain 501 mycelia, it was shown that the time from initiation to completion of infection with mycelia was much shorter (14 h) than with conidia (72 h). It was further shown that mycelial infection occurred predominately through direct penetration at intercellular junctions, while conidial infection occurred predominately through stomatal penetration. This suggests that mycelia are more suitable as infection propagules for A. alternata strain 501 in a bioherbicide for the control of E. adenophorum.     

Isolation, Pathogenicity and Safety of Curvularia eragrostidis Isolate QZ-2000 as a Bioherbicide Agent for Large Crabgrass (Digitaria sanguinalis)

A pathogen isolated from lesions on blighted leaves of crabgrass in three different locations of China was identified as Curvularia eragrostidis. Isolate QZ-2000 was the most virulent of six isolates tested. Experiments on morphology, pathogenicity, effect of environmental factors, and host-range of isolate QZ-2000 were conducted in the laboratory, greenhouse and field to assess the potential of this isolate as a biocontrol agent for grassy weeds. Pathogenicity was quantitatively determined based on mortality and dry-weight reduction of infected large crabgrass. Inoculum concentration, rapeseed oil concentration in formulaton, post-inoculation dew temperature and duration, and plant growth age all significantly influenced the efficacy of the isolate. A total of 85-100% control of large crabgrass was obtained when inoculum concentrations were ≥1×10⁶ conidia mL^-1, oil concentrations ≥0.9% (v/v), dew period ≥24 h and air temperatures 20-30°C in the greenhouse. A total of 51 plant species in 20 families were screened against isolate QZ-2000 in host-range studies. Six other species of grassy weeds were susceptible to isolate QZ-2000, but no mortality or significant dry-weight reduction was observed for maize (Zea mays), rice (Oryza sativa), soya bean (Glycine max), cotton (Gossypium hirsutum), or any other economically important crops and plants. In field trials, with 5×10⁶ conidia mL^-1 inoculum density, 60-7% reduction in dry weight was achieved for large crabgrass seedlings under natural dew-free conditions. These results indicate that isolate QZ-2000 is a potential microbial bioherbicide for control of large crabgrass in crops such as corn, soybean, cotton, water-melon, and peanut.     

Factors influencing biocontrol of jimsonweed (Datura stramonium L.) with the leaf-spotting fungus Alternaria crassa

In greenhouse tests, Alternaria crassa (Sacc.) Rands killed > 80% of inoculated jimsonweed (Datura stramonium L.) seedlings within 14 days following a 9-h dew period at 25°C with 1 × 10⁵ spores/ml, and after 8 h of dew at 1 × 10⁶ spores/ml. At least 10 h of dew with 1 × 10⁵ spores/ml and 9 h of dew with 1 × 10⁶ spores/ml were required to obtain 100% mortality of fungus-inoculated plants. Growth stage and inoculum concentration studies revealed that higher concentrations of inoculum were required to obtain 100% mortality of larger plants. Weed control was significantly reduced by day/night air temperatures of 35°C and 24°C, respectively, at all inoculum concentrations as compared to the controls at lower air and dew temperature regimes. The results of these studies indicate that A. crassa has potential as a biological herbicide for the control of jimsonweed.     

Drechslera sp. (Pyrenophora chaetomioides (Speg.)), a Potential Biocontrol Agent for Bromus sterilis and Other Bromus spp.

Infection by a Drechslera sp. (perfect stage, Pyrenophora chaetomioides (Speg.)), isolated from Bromus sterilis , killed B. sterilis , B. commutatus and B. secalinus. B. diandrus and B. hordeaceus were both infected but not killed. Successful infection required a 24-h dew period. Reduction of the dew period to 8 h significantly reduced the infection of all Bromus spp. tested as determined by leaf necrosis. Inoculation with a low inoculum concentration (2 104 conidia/ml) produced little dry weight reduction, but at 2 105 conidia/ml with an 8-h dew period the dry weights of B. commutatus , B. diandrus , B. secalinus and B. sterilis were reduced by 11-25%. Extending the dew period to 24 h resulted in 77% mortality of B. sterilis and 93% mortality of B. commutatus and B. secalinus.     

Factors Influencing the Efficacy of the Potential Microbial Herbicide Alternaria alternata (Fr.) Keissler on Amaranthus retroflexus (L.)

Alternaria alternata , applied to run-off at 107 conidia ml -1 and given a 6 h dew period, caused an 88.5% reduction in dry weight of 4 to 5 true-leaf plants of Amaranthus retroflexus . This level of control was achieved whether the inoculated plants were grown in the glasshouse or outside. Formulation of the pathogen in oilseed rape emulsion reduced its dew period requirement from 24 h or more to 8 h or less. For example, with 106 conidia ml -1 , equivalent effects were found with 8 h dew with emulsion formulation compared to 48 h dew with the Tween formulation. Allowing spray deposits to dry on inoculated plants for ca 7 h, before exposing to dew increased the efficacy of the pathogen.     

裸脚菇0612-9液体菌种优化及其对后续发酵产活性物质的影响

裸脚菇0612-9次级代谢产物具有强烈抗青绿霉活性,可作为微生物源防腐剂用于柑橘保藏,但是其发酵周期长,产出能耗大效率低。用摇瓶对裸脚菇0612-9的液体菌种培养基、培养条件进行优化并对优化后液体菌种接种种龄、接种量进行探索,最后用5L发酵罐进行放大发酵验证。取样计数测定菌丝球数量、过滤称重测定菌丝干重、HPLC监测活性物质Ⅱ的积累、牛津杯法评价抗青绿霉活性。经研究最佳碳源为玉米粉和麦芽糖,最佳氮源为蛋白胨,最佳液体菌种培养基组成为：玉米粉30g/L、麦芽糖10g/L、蛋白胨15g/L、KH₂PO₄ 2g/L、MgSO₄·7H₂O 1g/L;最佳培养条件：起始pH 5、接种3×Ф7mm菌块、装液量100mL/250mL三角瓶、温度28℃、转速160r/min;优化前菌丝球数46个/10mL,菌丝干重0.28g/100mL,优化后菌球数达985个/10mL,菌丝干重达0.69g/100mL,分别为优化前的21.4倍、2.43倍;后续发酵使用种龄9d的液体菌种、接种量7.5%。优化后液体菌种在发酵罐中后续发酵周期从10d缩短至5d,缩短50%,产量比优化前提高8.28%。     

Myrothecium verrucaria isolates and formulations as bioherbicide agents for kudzu

The fungus Myrothecium verrucaria (MV) has previously been shown to have potential as a bioherbicide for kudzu (Pueraria lobata) control. It has also been shown that MV wild-type (MV-wt) often forms sectors, when grown on various nutrient media. Experiments compared MV-wt and MV sector efficacy when grown on agar or on rice grains. In greenhouse evaluations of sectors, applied as foliar sprays in water or in other formulations (corn oil, surfactant, and corn oil plus surfactant) for efficacy against kudzu seedlings, some sectors possessed bioherbicidal activity equal that of MV-wt, but others exhibited lower activity. Without a dew period, aqueous formulations of MV-wt, a yellow sector, and a white sector provided zero control, but all three isolates were active without a dew period when formulated in corn oil, Silwet L-77 surfactant, and in surfactant plus corn oil. Generally, the yellow sector was less effective than the other two isolates in any formulations, and the MV-wt and white sector provided approximately 100% mortality of the test plants. Dew (10 h) increased weed control to 100, 33, and 65%, respectively, for MV-wt, the yellow sector and the white sector. All isolates provided nearly 100% control in the oil and surfactant formulations with a dew period compared to treatments receiving no dew. Soil incorporation studies were also performed to compare MV-wt efficacy of preparations grown on agar versus growth on rice grains. Higher efficacies (1.75-3.3-fold increase) were obtained from rice grain preparations compared to preparations grown on agar, when preparations were incorporated at several rates into soil prior to planting. Cell-free extracts of the MV-rice cultures were also phytotoxic to kudzu seedlings up to the eight- to 10-leaf growth stage. Thus, formulation, growth media, and the application method are important determinants in the efficacy of MV and MV sectors on kudzu seedlings.     

Efficacy of Trematophoma lignicola Petrak as a Biological Control Agent for Amaranthus retroflexus L.

The pathogenicity of Trematophoma lignicola on Amaranthus retroflexus was increased when its conidia were formulated in oilseed rape emulsion. However, this formulation did not reduce the dew period required to allow conidial germination and infection of the host, nor did it protect the conidia from desiccation before the onset of dew. The fungus gave effective control of A. retroflexus plants up to the 4-true-leaf stage. Plants with 5 to 6-true-leaves were infected and their dry weight reduced, but plants with more than 6-true-leaves were unaffected. Plants grown in warm conditions (18-21°C/12-15°C, day/night) were more likely to be successfully controlled than those grown in the cold (10-12°C/7-8°C; day/night). Significant dry weight loss of A. retroflexus plants were achieved at application volumes down to 100 l ha -1 at 5 ×10 6 conidia ml -1 . More effective control was achieved by application of conidia of T. lignicola than mycelial or pycnidial applications.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

Effect of inoculum density, stage of plant growth and dew period on the incidence of black point caused by Alternaria alternata in durum wheat

Glasshouse studies showed that the incidence of black point caused by Alternaria alternata in durum wheat was positively correlated with both the density of the inoculum and the growth stage of the wheat plants at the time of inoculation. A curvilinear relationship of the form Y=a + log X was found between inoculum density and disease incidence. The incidence of black point was linearly related to the stage of plant growth, between anthesis and the late milk stages of development, at the time of inoculation. A better relationship between growth stage and disease incidence was found when plant growth was expressed as days after anthesis than when the Romig scale was used (R²= 0.30 and 0.24 respectively). A threshold dew period of 3–6 h was required for black point symptoms to develop. The incidence of black point increased with increasing duration of the dew period until maximum disease incidence occurred after exposure to a dew period of 48 h.     

胶孢炭疽菌婆婆纳专化型侵染波斯婆婆纳的影响因子的研究

通过盆栽试验,研究了真菌自身生物学特性、杂草生长状况和环境因素等对胶孢炭疽菌婆婆纳专化型菌株QZ－97a侵染波斯婆婆纳的影响。结果表明,子叶期和衰老斯波斯婆婆纳感病性最强;培养7－14d后的孢子侵染力最强;病害发生的最佳温度范围是15－25℃;在保湿2－3d的条件下,露期中间的光照时间越短,病害发生越严重;保湿也可通过添加玉米粉和黄粉等介质来解决;达到理想致病效果的该菌株孢子悬浮液浓度必须在10^7个.ml^-1以上,由此可见,菌株QZ－97a在波斯婆婆纳发生时期的环境条件下可有效控制该杂草。     

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi is described. A comparison of conditions led to the following standard conditions being recommended. The first or second pair of leaves of seedling plants at the V8 growth stage are inoculated using inoculum grown on sunflower leaf extract agar for 5–10 days at an inoculum density of 1–2 spores cm² of leaf tissue. A 48 h dew period should be applied to plants covered by a plastic tent. A dew period temperature of 26/26°C night/day and a post-dew period temperature relative to that experienced under local growing conditions should be applied. Lesions are measured 7 days after inoculation, and mean lesion size per plant is calculated. Mean lesion size of lines being tested is expressed as a proportion of the mean lesion size of a susceptible standard included in each screening experiment.     

Biological Control of Kudzu ( Pueraria lobata ) with an Isolate of Myrothecium verrucaria

An isolate of the fungus Myrothecium verrucaria ( MV ) was evaluated for biocontrol potential against kudzu ( Pueraria lobata ). In greenhouse tests, MV was highly virulent against kudzu in the absence of dew when conidia were formulated in 0.2% Silwet L-77 surfactant (SW). Inoculum concentrations ≥2 ×10 7 conidia ml -1 were required to satisfactorily control plants in the third leaf stage and larger. In controlled environment experiments, kudzu mortality was greater at higher temperatures (25-40°C) than at lower temperatures (10-20°C), although pathogenesis and mortality occurred at all temperatures tested. In field tests, transplanted kudzu seedlings in the 2-3 leaf growth stage treated with MV at 2 ×10 7 conidia ml -1 in 0.2% SW, exhibited leaf and stem necrosis within 24 h following inoculation, with mortality occurring within 96 h. After 7 days, 100% of inoculated kudzu plants were killed in plots treated with the fungus/surfactant mixtures. Similar results were observed in a naturally occurring kudzu population, where 100% control occurred within 14 days after inoculation with 2 ×10 7 conidia ml -1 in 0.2% SW. In summary, MV effectively controlled kudzu in the absence of dew over a wide range of physical and environmental conditions and under field conditions. These results indicate that, when properly formulated, MV has potential as a valuable bioherbicide for controlling kudzu.     

Investigation of Spray Application of Microbial Herbicides Using Alternaria alternata on Amaranthus retroflexus

Inclusion of Alternaria alternata conidia in a spray formulation affected the distribution pattern on the target. The dry weight of Amaranthus retroflexus plants was reduced by more than 83% when A. alternata conidia (107 ml -1 ) were applied at 200 L ha -1 or greater and when given a 24 h dew period. At low application volumes (25 or 50 L ha -1 ) plant dry weight was reduced by only 29 or 54%. After 7-8 h dew period, conidial germination on the leaf surface was 11-19%. This increased to 62-91% after 24 h dew period. Counts of conidia on leaves indicated that up to 86% of the conidia sprayed were not retained on the target plant, or did not reach it. This is reflected in lesion numbers per unit area being only 3-5% of the calculated theoretical numbers. The results cast doubt on the suitability of A. alternata as a microbial herbicide for the control of Am. retroflexus .     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.     

木香薷精油提取最佳采收期的研究

采用测定生物量与精油提取相结合的方法来研究确定木香薷的最佳采收期。结果表明:霍山地区木香薷的最佳采收期在8月中旬。木香薷生物量的增长在前期主要表现为当年生小枝数的增加,而后期表现为当年生小枝重量的增加。从5月12日到8月17日,每100 g干重的当年生枝产精油量逐渐增加,而8月17日之后精油产量逐渐下降,以8月7日为最高达到1.62 g,得率为1.62%。     

水体酚类化合物污染对水稻幼苗生长的影响

用0、50、100和150 X 10-6浓度的苯酚溶液处理水稻幼苗,本实验的目的是探讨苯酚对水稻幼苗生长的影响。实验结果如下：苯酚抑制水稻幼苗生长,且随着浓度的增加,其抑制作用越明显。150 ppm是抑制作用最强的浓度。经苯酚处理后的水稻幼苗,其根和茎的生长都抑制了,而鲜重、干重、水分含量和叶绿素含量则明显减少,硝酸还原酶和过氧化物酶活性也下降了。     

Factors Affecting Disease Development by Rhynchosporium alismatis in Starfruit (Damasonium minus), a Weed of Rice

The fungal pathogen Rhynchosporium alismatis is being developed for biological control of starfruit (Damasonium minus), an important aquatic weed in Australian rice fields. The development of R. alismatis in starfruit differs between juvenile and adult plants. Juvenile starfruit plants are stunted as a result of fungal infection, while in adult plants, the main effect is necrosis and chlorosis of floating leaves. A conidial concentration of 1×10⁴ conidia mL^-1 was adequate to cause disease symptoms on floating leaves, but the stunting effect on juveniles was caused by concentrations of at least 1×10⁵ conidia mL^-1. To successfully inoculate juvenile plants, the water must be drained before inoculation to expose plants to the inoculum. The artificial addition of dew periods did not enhance disease development in plants. The stunting of juvenile starfruit plants caused by the infection of R. alismatis may give rice plants a competitive advantage over the weed at the seedling stage