Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Effect of relative humidity and origin of isolates of Neozygites tanajoae (Zygomycetes: Entomophthorales) on production of conidia from cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae), cadavers

Neozygites tanajoae is a very specialized fungus pathogenic to the cassava green mite (CGM), Mononychellus tanajoa, an important cassava pest introduced to Africa from the Neotropics. Conidial discharge from cadavers of CGM that died from infections with 14 isolates of N. tanajoae collected from diverse climates of Brazil was quantified to help select potential candidate strains for introduction to Africa. Studies aimed to identify isolates with lower requirements for relative humidity for sporulation and isolates that discharged more conidia during short periods of moisture. At 96 ± 0.5% RH, production of conidia was variable and even isolates from the Brazilian semi-arid region, e.g., Petrolina and Itaberaba, produced few conidia. Significant differences in the numbers of conidia produced by diverse Brazilian isolates were observed after 6, 9 and 12 h at 100% RH. At 100% RH, production of primary conidia increased considerably from an average of 57 ± 4 conidia at 6 h to 509 ± 37 conidia at 12 h. The isolate sporulating least (BIN21) discharged only 45.7% of the number of conidia produced by isolate BIN1, one of the isolates producing the most spores. Results from this study demonstrate that differences in production of conidia among isolates should be considered when selecting Neozygites isolates for new biological control introductions.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

A new endophytic species of Arthrinium (Apiosporaceae) from Jatropha podagrica

During study of endophytic fungal diversity in tropical region, a new species of Arthrinium was isolated from healthy petiole of Jatropha podagrica. It is characterized by aseptate, dark brown, circular, lenticular conidia with hyaline margin, broad, hyaline conidiophores with numerous brown septa and ampulliform–lageniform conidiophore mother cells. The new species superficially resembles A. phaeospermum and A. saccharicola in having circular lenticular conidia, but differs in conidia and conidiophore size, and shape of conidiophore mother cells. Phylogenetic study using sequence analysis of ITS region (ITS1–5.8S–ITS2) separates the new species (A. jatrophae) from other related species of Arthrinium.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

A distributed delay routine-based simulation model of Beauveria bassiana conidial stability in response to environmental stressors

Using published data and equations on therelationship between spore longevity of theentomopathogenic hyphomycetes, Metarhiziumanisopliae var. acridum and Beauveria bassiana (Balsamo) Vuillemin(Deuteromycota: Hyphomycetes) and temperatureand moisture content, a model of sporeviability was constructed based on adistributed-delay routine. The model ismodified via average spore survival time or byincluding an additional attrition (mortality)rate. The model was parameterized usingpublished values from studies on M. a.var. acridum spores, and output comparedfavorably with germination data and with apreviously-developed model. After initializingthe model using parameter estimates of B.bassiana spores from the laboratory andpublished data on changes in (1) spore viabilitywith respect to temperature and moisturecontent, and (2) spore moisture content withrespect to temperature and relative humidity,the model was run using daily min/maxtemperature and relative humidity data andcompared with data from four field experimentsof Mycotech B. bassiana isolate GHAsprayed on canteloupe plants. For two of theexperiments, observed viability trends werecompared to model outputs using weather datafrom both a weather station and fromwithin-canopy temperature and humidity probes. Output using weather station data fitobservations much better than output usingwithin-canopy probe data. For the tworemaining sets of field data, both earlier inthe season, only weather station data wereavailable and the resulting output fitobservations poorly. An attrition rate of 98%was needed to fit output to field data early inthe growing season, and a rate of 74% wasneeded for data collected four weeks later. These attrition rates can be consideredestimates for the proportion of spores dyingfor reasons other than temperature and relativehumidity, and they were attributed largely toUVB radiation due to the more open canopyearlier in the season.     

培养基及培养条件对冬虫夏草菌固体发酵产分生孢子的影响

为获得冬虫夏草菌固体发酵产分生孢子的最优工艺,以野生分离的冬虫夏草菌为材料,对其固体发酵产分生孢子的培养基及培养条件进行了研究。试验结果表明：泥炭土为最佳基础培养基,该培养基中冬虫夏草菌气生菌丝生长一般,但产分生孢子最多,可达4.2×10³个/g;泥炭土培养基中添加0.1‰ IAA（吲哚乙酸）、0.1‰ IBA（吲哚丁酸）和0.1‰ NAA（萘乙酸）能促进冬虫夏草菌气生菌丝的生长和分生孢子的产生,其分生孢子达8.1×10³个/g;该基础培养基中,冬虫夏草菌于18℃培养30d后,在10℃、相对湿度45%、蓝光照射进行诱导,分生孢子可达1.0×10⁴个/g。本研究建立了一种大量获取冬虫夏草菌分生孢子的方法,为冬虫夏草繁育奠定了基础。     

Environmental factors impact temporal Passalora sequoiae conidia counts from Leyland cypress

Needle blight disease, caused by Passalora sequoiae, results in a progressive loss of leaf tissue on Leyland cypress (×Cupressocyparis leylandii) within container and field tree nurseries, in the landscape and on Christmas tree farms. Fungicide schedules have been developed in response to seasonal symptom progression and conidia dispersion without clarification of the influence of weather conditions on disease. Conidia count data of P. sequoiae were collected approximately weekly from June to December in 2001 in Watkinsville, GA, and in 2002 in Dearing, GA. Peak conidia numbers were trapped in September and October. Daily weather data summaries were obtained from a weather station at each location. Deterministic models were developed individually for summer (1 June to 30 September) and fall (1 October to 21 December) seasons. A three‐day moving average (MA) of average daily temperature (avgT) had a positive quadratic relationship with conidia counts in the summer season. A 10‐day MA of avgT, vapour pressure deficit (VPD) and total solar radiation (SolR) had a positive quadratic, a negative linear and positive linear relationship, respectively, with conidia counts in the fall season. The models show that the seasonal shift to cooler fall temperatures, along with lower VPD and SolR, favour production and dispersal of P. sequoiae conidia.     

纯培养下拟茎点霉分生孢子的形成及意义

以18种共23个拟茎点霉菌株为材料,比较了在25℃、12h光照(40w日光灯)/d条件下不同培养基上甲、乙型分生孢子的形成情况。结果表明,苜蓿煎汁+Czapek培养基能促使多数拟茎点霉菌株形成甲、乙两型分生孢子,其形态及大小也与自然寄主上的相一致。根据纯培养获得的形态特征对原始描述缺乙型分生孢子特征记载的Phomopsis durionis, homopsis sterculicola, Phomopsis macadamii, Phomopsis lucumicola和Phomopsis tinea进行了补充描述。     

Effects of a biocontrol agent of apple powdery mildew (Podosphaera leucotricha) on the host plant and on non-target organisms: an insect pest (Cydia pomonella) and a pathogen (Venturia inaequalis)

A number of studies have focused on the selection and use of new biocontrol agents, but the effects of the introduction of these microorganisms on non-target organisms, including the crop plants themselves, are not well known. Non-target effects of sprayed applications of a potential biocontrol agent of apple powdery mildew (Podosphaera leucotricha Ell. Et Ev.), on scab infections (Venturia inaequalis Cooke Winter), on codling moth [Cydia pomonella L. (Lepidoptera: Tortricidae)] oviposition and damage and apple (Malus x domestica) fruit quality are examined. This biocontrol agent, an epiphytic yeast isolate called Y16, affected neither conidia germination of V. inaequalis nor their penetration of the leaf tissue but suppressed the disease caused by this pathogen. The quantity of eggs laid by the codling moth during its second flight period on yeast treated trees was significantly different to the quantity of eggs laid on the untreated trees. In the first season of the experiments, more eggs were laid on the treated trees, especially on those tree parts closest to the fruit. These results, however, were not confirmed the following season: fewer eggs were laid on the treated trees than on the untreated trees. These conflicting observations are attributed to year-to-year variation in environmental conditions, which may affect yeast survival and activity. A 2-month-long assay was conducted in the orchard during the codling moth's second flight period from mid-July until mid-September. The yeast treatment did not affect the damage caused by the codling moth to the fruits. Finally, the yeast treatment did not affect any of the examined fruit quality parameters.