Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

Pectinase and cellulase enhance the control of Abutilon theophrasti by Colletotrichum coccodes

Inundative mycoherbicidal biocontrol agents are typically insufficiently virulent to be commercially competitive with herbicides in row crop agriculture, and require enhancement. Pectinase and cellulase are typically used by pathogens during infection. Thus, it was hypothesized that adding exogenous cell wall degrading enzymes might enhance fungal infection. Pectinase or cellulase was added to inocula of aqueous chopped mycelial suspensions of a strain of Colletotrichum coccodes for control of Abutilon theophrasti. Plants treated with 5.3×10⁶ C. coccodes propagules mL^-1 and 1.65 U mL^-1 pectinase had more rapid and complete disease development. Similar trend was achieved when 10 U mL^-1 of cellulase were added to 2.2×10⁶ C. coccodes propagules mL^-1. Adding pectinase or cellulase did not increase the host range of the wild-type fungus. The results suggest that there might be value to transforming biocontrol agents to overproduce these enzymes.     

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10¹ and 1.9 × 10⁴ cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.     

Colletotrichum sp: A potential candidate for biocontrol of scentless chamomile (Matricaria perforata) in western Canada

Based on an assessment of 706 fungal isolates obtained from Canada and Europe, a group of Colletotrichum sp. isolates, tentatively identified as C. truncatum, was moderately efficacious for biocontrol of scentless chamomile (Matricaria perforata). In this study, 19 C. truncatum isolates, 11 from Canada and eight from Europe, were compared for virulence, crop safety, and minimum dew requirement for infection to narrow the selection of candidates. Applied at 1×10⁶ spores mL^-1, these isolates expressed variable virulence under controlled environments, with slightly higher variations observed on the Canadian isolates. There was also a slight difference in host specificity among the isolates tested; most isolates caused disease only on chamomile species (M. perforata and M. recutita) but two Canadian isolates also infected lentil, flax, or both. At 20°C, most isolates required more than 20 h dew for maximum infection. This requirement can be an impediment for using this fungus as a biocontrol agent in western Canada where the climate is semi-arid. Treatment of scentless chamomile at the 10-leaf stage with the herbicide metribuzin 48 h prior to fungal inoculation increased weed control to 72%, compared to 40 and 47% by the herbicide and fungus applied alone. However, a similar treatment using the herbicide bentazon did not enhance the weed control significantly as compared to the herbicide alone.     

Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode.

An enzyme-linked immunoassay coupled with a tyrosinase modified enzyme electrode was used for rapid detection of Campylobacter jejuni. The immunomagnetic separation (IMS) method was investigated to achieve optimal isolation of C. jejuni cells. Eight types of beads with three different sizes and function groups were coated with anti-C. jejuni to isolate C. jejuni from the sample solution. Bead size and coating methods were found to be major factors that influenced the capture efficacy. Streptavidin-labeled beads (2.8 μm) provided the greatest capture ability. Three blocking reagents were tested to minimize non-specific binding. Bovine serum albumin (BSA) showed the best blocking capability. Two IMS formats were tested. Competitive immunoassay cut the detection time to 1.5 h, but the detection limit was relatively high (10⁶ CFU/ml). This system was evaluated using C. jejuni pure culture and poultry samples inoculated with C. jejuni. This detection method for C. jejuni could be completed within 2.5 h and had a detection limit of 2.1×10⁴ CFU/ml. No significant difference was found between pure culture samples and poultry samples (P>0.01). A linear relationship was found between C. jejuni cell numbers and the peak current ratio in a range of 10²–10⁷ CFU/ml (R²=0.94).     

热带、亚热带典型森林-土壤系统植硅体碳演变规律

分别选取中国亚热带毛竹林、马尾松林、青冈林、杉木林和热带青梅林、芭蕉林、橡胶林、马占相思林8种森林类型,采集其鲜叶、凋落叶以及0～10和10～30 cm土层土壤,通过微波消解法提取其中的植硅体,并采用碱溶法测定植硅体中碳含量.结果表明: 4种亚热带森林类型鲜叶、凋落叶和0～10 cm土层中植硅体碳含量均以马尾松林(230.24、229.17、20.87 g·kg^-1)最高,毛竹林(30.55、37.37、3.38 g·kg^-1)最低,10～30 cm土层则以青冈林(18.54 g·kg^-1)最高,毛竹林(2.90 g·kg^-1)最低.热带森林鲜叶中植硅体碳含量以马占相思林(377.66 g·kg^-1)最高,青梅林(46.83 g·kg^-1)最低,凋落叶中则是橡胶林(218.23 g·kg^-1)最高,芭蕉林(27.66 g·kg^-1)最低,而0～10和10～30 cm土层土壤中均以马占相思林(23.84、24.90 g·kg^-1)最高,芭蕉林(3.89、3.93 g·kg^-1)最低.与0～10 cm表层土相比,杉木林、青冈林、马尾松林、毛竹林、橡胶林、马占相思林、芭蕉林和青梅林鲜叶植硅体碳含量分别下降97.4%、94.9%、90.9%、88.9%、95.9%、93.7%、93.3%和63.7%.青冈林、芭蕉林和马占相思林鲜叶植硅体碳含量显著高于凋落叶,而毛竹林、马尾松林、杉木林、青梅林和橡胶林之间无显著差异.8种森林类型土壤植硅体碳含量均显著低于鲜叶和凋落叶,表明植硅体在通过凋落物释放到土壤的过程中是不稳定的.     

巴茅草水浸提液对3种作物种子萌发及幼苗生长的化感作用

本文以发芽率、发芽速度指数、发芽指数、根长、茎长和生物量为种子萌发和幼苗生长参数,研究不同生长时期巴茅草叶片和茎秆水浸提液对白菜、生菜、水稻的化感作用。结果表明: 巴茅草叶水浸提液化感作用强于茎秆水浸提液,叶水浸提液处理后受体植物的发芽指数和生物量均显著低于茎水浸提液。枯萎期巴茅草的化感作用强于生长旺盛期。不同浓度巴茅草叶水浸提液对3种作物的化感作用存在明显的量效关系,浸提液浓度越高,巴茅草的化感抑制作用越强。巴茅草叶水浸提液对白菜和生菜各萌发指标100%抑制的浓度分别为0.075和0.10 g·mL^-1;而0.10 g·mL^-1巴茅草叶水浸提液对水稻发芽率、发芽速度指数、发芽指数的抑制率分别为13.8%、27.2%、19.3%。巴茅草叶水浸提液对白菜和生菜各生长指标100%抑制的浓度分别为0.05和0.10 g·mL^-1;而0.10 g·mL^-1巴茅草叶水浸提液对水稻根长、茎长、生物量的抑制率分别为64.6%、92.9%、21.8%。结合种子萌发和幼苗生长的综合化感指数,3种供试作物对巴茅草化感作用的敏感程度为白菜>生菜>水稻。     

凋落叶多样性对杉木幼苗生长及吸收15N标记硫铵的影响

利用¹⁵N硫铵研究了凋落叶多样性对杉木幼苗生长及养分吸收的影响.结果表明,凋落叶多样性的增加有利于盆栽杉木幼苗的生长.杉木、火力楠、红栲和刺楸4种凋落叶混合处理后,杉木幼苗的生长量最大;杉木、火力楠、刺楸3种凋落叶混合处理后的杉木幼苗生长量次之,其它依次为杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>对照>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.就杉木幼苗对硫铵氮的吸收率而言,不作任何处理的杉木幼苗吸收率最高,其次为杉木、火力楠、红栲和刺楸4种凋落叶混合处理,其它依次为杉木、火力楠、刺楸3种凋落叶混合处理和杉木、火力楠、红栲3种凋落叶混合处理>杉木和刺楸凋落叶处理>杉木和红栲凋落叶处理>杉木和火力楠2种凋落叶混合处理>杉木凋落叶处理.另外,用凋落叶处理后,土壤中硫铵氮的残留量比不作凋落叶处理的土壤多,硫铵氮的总回收量也比不作凋落叶处理的土壤大幅增加,而且凋落叶多样性的增加也会增加硫铵氮的残留量.     

Efficacy of the Entomopathogenic Fungus, Culicinomyces clavisporus against Larvae of the Biting Midge, Culicoides nubeculosus (Diptera: Ceratopogonidae)

Culicinomyces clavisporus, a fungal pathogen of a wide range of mosquito species, was investigated in relation to potential pathogenicity against Culicoides nubeculosus biting midge larvae. Seven different C. clavisporus strains were assayed. Each showed some degree of activity against C. nubeculosus larvae with LC₅₀ values of between 3.2×10^-5 and 1.1×10^-6 spores/mL; these effects occurred in dose-dependent manners and tended to be delayed until 72-96 h post treatment. The results are discussed in relation to incorporation of C. clavisporus into biocontrol programmes for Culicoides spp.     

Ultrastructural study of developmental stages of Mattesia dispora (Neogregarinorida: Lipotrophidae), a parasite of the flour moth Ephestia kuehniella (Lepidoptera)

The ultrastructure of merozoites, gamonts and oocysts of the neogregarine Mattesia dispora and their development in larvae of the flour moth Ephestia kuehniella were studied by electron microscopy. The apical complex of free macronuclear merozoites was very distinct in micrographs of sections, the polar rings being especially prominent. Two gamonts associated in head-to-head syzygy and the apical complexes served as the contact point during pairing. At this stage the rhoptries became reduced and the conoid widened. The gamonts had a foam-like appearance in the light microscope. Paired gamonts formed an envelope and developed into a gametocyst, within which the gamonts were separated by a distinct border. Four gametes and two residual cells developed inside the gametocyst. The gametes were covered with a single membrane. The gametes fused in pairs to form two spherical zygotes, each covered by two membranes and with one large nucleus. The external layer appeared more undulated than the inner one. A single membrane covered each residual cell. Walls were formed around both zygotes to produce two oocysts. Each mature oocyst was lemon-shaped with polar plugs and eight peripheral sporozoites, which had a pellicle similar to that of the merozoites, lay beneath the thick oocyst wall.     

Evaluation of the bioherbicide Myrothecium verrucaria for weed control in tomato (Lycopersicon esculentum)

An isolate of the fungus Myrothecium verrucaria was evaluated for its biocontrol potential against common purslane, horse purslane, spotted spurge, and prostrate spurge, all serious weed pests in commercial tomato fields in the southeastern US. In greenhouse and field tests, M. verrucaria was highly virulent against these weeds when applied as conidial sprays formulated in 0.2% Silwet L-77 surfactant, even in the absence of dew. In field test plots naturally infested with these weeds, seedlings in the two-to-three leaf growth stage treated with M. verrucaria at 2×10⁷ conidia mL^-1 in 0.2% Silwet, exhibited leaf and stem necrosis within 24 h following inoculation, with mortality occurring within 96 h. After 7 days, M. verrucaria had killed 90-95% of both purslane species and 85-95% of both spurge species. Tomatoes that were transplanted into plots treated with M. verrucaria remained healthy and vigorous throughout the growing season. Since M. verrucaria effectively controlled several common weeds under field conditions, this fungus appears to have potential as an effective bioherbicide for pre-plant weed control in production systems with transplanted tomato.     

Critical thermal maximum and body water loss in first instar larvae of three Cetoniidae species (Coleoptera)

Critical thermal maximum (CT_max) and body water losses were measured in first instar larvae of Gnorimus nobilis, Osmoderma eremita (Trichiinae) and Cetonischema aeruginosa (Cetoniinae) when air temperature was increased gradually (0.5 °C/min) from 20 °C to the critical thermal maximum (CT_max), in dry air (near 0% R.H.).

The CT_max was significantly lower in O. eremita (45.6±0.7 °C) than in G. nobilis (48.5±0.6) and C. aeruginosa (51.4±0.9 °C).

An increase of 10 °C (30–40 °C) induced a 2-fold increase of the water loss in C. aeruginosa and O. eremita (Q₁₀=2.10±0.12 and 2.13±0.20, respectively). In the range from 40 to 45 °C to CT_max a strong increase of the water loss was observed in O. eremita and C. aeruginosa, respectively. Body water losses were significantly lower in C. aeruginosa than in O. eremita and G. nobilis over the range 20 °C—CT_max; no significant difference occurred between G. nobilis and O. eremita.     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

α-Chymotrypsin shows higher activity in water as well as organic solvents after three phase partitioning

Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The k_cat/K_m of the partitioned enzyme (TPP-C) for hydrolysis of Bz-Tyr-OEt in aqueous medium at 25°C was found to be 48.3×10⁴ mM^-1 min^-1 as compared to the corresponding value of 17.7×10⁴ mM^-1 min^-1 for the untreated control (C). The λ_max of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v^-1) water (in t-amyl alcohol), while no esterification was observed with C, TPP-C still showed significant level of esterification activity.     

Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae

Since the collapse of populations of northern quahogs (hard clam), Mercenaria mercenaria, in Long Island bays, brown tide blooms have been proposed to pose a barrier to recovery. We tested whether the brown tide alga, Aureococcus anophagefferens, affects survivorship, development or growth in the larvae of M. mercenaria. There was no effect of A. anophagefferens (clone CCMP1708) on survivorship of hard clam larvae, even at bloom concentrations. Under most experimental conditions, larvae fed a mixed diet of Isochrysis galbana (T-Iso) and A. anophagefferens or a single species diet of A. anophagefferens, developed faster than those fed a single species diet of Isochrysis. A mixed diet of I. galbana and A. anophagefferens either had no effect on larval growth, or produced enhanced growth at moderate cell densities (8 × 10⁴ cells ml⁻¹ of A. anophagefferens). Similarly, moderate cell densities of a single food diet of A. anophagefferens (1.6 × 10⁵ cells ml⁻¹) generally had no effect on the growth of larvae. When fed bloom concentrations (10⁶ cells ml⁻¹) of A. anophagefferens, larvae developed faster, but growth was reduced, compared to those fed an equal biovolume of Isochrysis. Larvae fed slow growing or near stationary phase cultures of A. anophagefferens experienced reduced growth and slowed development. These data suggest a qualitative difference between slow or stationary phase and fast growing cultures of the brown tide alga. They also suggest that impacts of A. anophagefferens, when present, are likely to be due to the nutritional quality of this alga as a food source for hard clam larvae, which could have a lasting legacy through ontogeny. Additional studies are needed to test whether our findings apply to more recently isolated strains of A. anophagefferens.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation

Experiments were carried out to evaluate the influence of rearing temperature and food concentration (20 and 30 °C, 1×10⁵ and 2×10⁵ cells ml⁻¹) on the starvation threshold and nucleic acid content of the larvae of Balanus amphitrite. The larvae were also field-reared using micro-enclosures. Laboratory-reared larvae were larger in size than the field-reared larvae. An increase in size, DNA content and instar index of the starved II instar larvae was observed indicating that the absence of food may not be fatal to this early instar. The temperature at which larvae were raised and the food concentration had variable influence on the capacity to withstand starvation. Exposure to increased temperatures during starvation eliminated the effect of doubling food concentration during their feeding period prior to starvation. The larvae reared at 20 °C had comparatively lower nucleic acid content. The laboratory-reared larvae had ca. 1.7 times greater RNA:DNA ratio than larvae raised at comparable temperature in the field.     

A model for the involvement of Okazaki fragments maturation in the expansion of short tandem repeats

Mutations were accumulated with a wide variety in the p53 pseudogene of various wild mouse species and subspecies captured at different localities, as extensively observed in the exon 4 – exon 5 region. The rate of mutation accumulation in the mouse p53 pseudogene was estimated to be 1.4–2.1×10⁻⁸ mutations/bp/year, which is 20–30 times faster than that of the functional p53 and makes the dating possible for the time range of 10⁶ years or more. From comparison of the mutation spectrum, the origin of laboratory mice was identified to one of two M. m. domesticus groups.     

Lack of effect of the nematophagous fungus Duddingtonia flagrans on the development of the dung beetle, Aphodius constans

The nematode-trapping fungus Duddingtonia flagrans may be used as a biological control agent of gastro-intestinal nematode larvae of ruminants by feeding the hosts with fungal spores. This trial was intended to search an eventual detrimental impact of the presence of spores of D. flagrans in high numbers in goat feces on the common dung beetle, Aphodius constans (Coleoptera: Aphodiidae). A. constans eggs were settled in feces derived from grazing goats fed spores at daily dose rates of 0, 0.25 × 10⁶, 0.5 × 10⁶ or 10⁶ spores/kg BW. At the end of the incubation period, the number of adults that have emerged from eggs were counted and compared between dose rates. No difference in emergence rate between treatments can be seen. The presence of D. flagrans spores in goat feces, even in large numbers, did not alter the development of A. constans.     

风车草(Cyperus alternifolius)人工湿地系统氮去除及氮转化细菌研究

研究了风车草人工湿地污水处理系统TN去除率及氮转化细菌的数量。结果表明:风车草人工湿地对TN的去除率为73.8%,与无植物人工湿地系统相比较,去除率提高了17.4%。风车草人工湿地氨化细菌为7.98×10⁵cfu·g^-1(细砂),硝化细菌为1.95×10⁵MPN·g^-1(细砂),反硝化细菌为5.89×10⁴1.95×10⁴MPN·g^-1(细砂)。与无植物系统氮转化细菌相比,氨化细菌无明显差异,硝化细菌及反硝化细菌均高出1个数量级。