Effect of temperature,humidity and photoperiod on mortality of Mononychellus tanajoa (Acari: Tetranychidae) infected by Neozygites cf. floridana (Zygomycetes: Entomophthorales)

The effect of temperature, humidity and photoperiod on the development of Neozygites cf. floridana (Weiser and Muma) in the cassava green mite, Mononychellus tanajoa (Bondar) was studied in the laboratory. Dead infected mites began to appear 2.5 days after inoculation. At 33 and 28°C peak mortalities were higher and occurred earlier (after 2.5 days), than at 23 and 18°C. Mean LT₅₀ (time for half the infected mites to die) decreased with increasing temperature as follows: 3.9, 3.0, 2.9 and 2.5 days at 18, 23, 28 and 33°C, respectively. When placed under conditions of high relative humidity for a period of 24 h, the percentage of dead infected mites from which the fungus sporulated was highest at 28°C (51.4%) and lowest at 33°C (6.5%). The development of the fungus inside the mite was not significantly affected by ambient humidity or photoperiod. No significant interactions between tested factors were found.     

A new species of <Emphasis Type="Italic">Discostroma</Emphasis> and its anamorph <Emphasis Type="Italic">Seimatosporium</Emphasis> with two morphological types of conidia,isolated from the stems of <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Conidia of two morphologically different types, one with a basal appendage only and the other with appendage at both ends, were isolated from the stems of Paeonia suffruticosa. Single conidial isolates of both types of conidia yield identical colonies, which then produced both types of conidia on agar media depending on temperature, thus showing that both types of conidia belong to the same fungus. Seimatosporium botan is described based on its morphological characteristics. The teleomorph of the fungus was first found on sterilized P. suffruticosa stems placed on water agar, when grown at 5°C for 2 months in 12-h photoperiod. Discostroma botan is described for this fungus. The teleomorph is also found on the same host in the field.     

Drying and formulation of blastospores of Paecilomyces fumosoroseus (Hyphomycetes) produced in two different liquid media

Formulation matrices can play an important role in improving the storage survival and biocontrol efficacy of microorganisms used for the control of pest insects. In this study, liquid culture-produced blastospores of the entomopathogenic fungus Paecilomyces fumosoroseus were formulated with different inert and organic materials prior to air-drying. Paecilomyces fumosoroseus blastospores were produced in two different liquid media, a basal salts medium supplemented with Casamino acids and glucose (LM1) and a medium containing peptone of collagen and glucose (LM2). Blastospores produced in the two test media were formulated with various supports. The formulation supports were cornstarch, rice flour, talc powders, Mexican lime, calcined kaolin clay, and diatomaceous earth. Several of the supports were tested at different concentrations. The initial and long-term (after storage at 4 and 28 °C) survival of the formulated, air-dried blastospores were evaluated. Initial blastospore viabilities were affected by the formulation material and by the blastospore production medium. Medium composition, drying support and storage temperature had an impact on the long-term survival of the blastospores. Under the conditions of the study, LM1 produced higher concentrations of blastospores that not only survived drying better than blastospores produced in LM2 but also maintained viability longer during storage in the formulation supports tested. The nature of the drying supports was shown to have a significant impact on the storage stability of all blastospores, particularly those produced in LM1. Under the production, drying and storage conditions used in the study, calcined kaolin clay formulations stored at 4 °C had the best storage stability. In all formulations tested, spore survival over time was reduced for blastospore formulations stored at 28 °C rather than 4 °C.     

Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies

The effect of temperatureon conidial germination, mycelial growth, andsusceptibility of adults of three tephritidfruit flies, Ceratitis capitata(Wiedemann), C. fasciventris (Bezzi) andC. cosyra (Walker) to six isolatesof Metarhizium anisopliae were studied inthe laboratory. There were significantdifferences among the isolates in the effect oftemperature on both germination and growth.Over 80% of conidia germinated at 20, 25 and30°C, while between 26 and 67% conidiagerminated at 35°C and less than 10% at15°C within 24 hours. Radial growth was slowat 15°C and 35°C with all of theisolates. The optimum temperature forgermination and mycelial growth was 25°C. Mortality caused by the six fungal isolatesagainst the three fruit fly species varied withtemperature, isolate, and fruit fly species.Fungal isolates were more effective at 25, 30and 35°C than at 20°C. The LT₉₀values decreased with increasing temperature upto the optimum temperature of 30°C. Therewere significant differences in susceptibilitybetween fly species to fungal infection at allthe temperatures tested.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Effects of pre-storage conditions on storage of in vitro cultures of Nephrolepis exaltata (L.) Schott and Cordyline fruticosa (L.) A. Chev.

In vitro cultures of Nephrolepis exaltata and Cordyline fruticosa were stored at 5°, 9° or 13°C, at a low irradiance (3–5 mol m^–2 s^–1) or in darkness. Prior to storage the cultures were subjected to 18°, 21°, 24° or 27°C and 15, 30 or 45 mol m^–2 s^–1 in a factorial combination.The optimal storage conditions for Nephrolepis were 9°C in complete darkness. These cultures were still transferable to a peat/perlite mixture at the end of the experimental period of 36 months.The optimal storage conditions for Cordyline were 13°C and a low light level (±3–5 mol m^-2 s^-1). When the pre-storage conditions were normal growth room conditions (24°C and 30 mol m^-2 s^-1), in vitro cultures could be stored for 18 months. With the most favourable pre-storage treatment (18°C and 15 mol m^-2 s^-1) some cultures still had green shoots after 36 months of storage, but did not survive transfer to peat/perlite.Pre-conditioning before storage was most favourable for Nephrolepis, and not that important, but still favourable, for Cordyline. There was an interaction between pre-storage temperature and pre-storage irradiance. For both species a high irradiance level was less favourable than a low irradiance level when combined with high growth room temperatures.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - NOA 2-naphthoxyacetic acid     

Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica

In order to effectively preserve green spores, which have relatively higher water content and lose viability more quickly than non-green spores, we studied the effect of desiccation level and storage temperature on Osmunda japonica spores. The water content of fresh spores was 11.20%. After 12 h desiccation by silica gel, the water content decreased to 6% but spore viability did not change significantly. As the desiccation continued, the decrease in water content slowed, but spore viability dropped. For almost all storage periods, the effects of storage temperature, desiccation level, and temperature × desiccation level were significantly different. After seven days of storage, spores at any desiccation level stored at 4 °C obtained high germination rates. After more than seven days storage, liquid nitrogen (LN) storage obtained the best results. Storage at −18 °C led to the lowest germination rates. Spores stored at room temperature and −18 °C all died within three months. For storage at 4 °C and in LN, spores desiccated 12 and 36 h obtained better results. Spores without desiccation had the highest germination rates after being stored at room temperature, but suffered the greatest loss after storage at −18 °C. These results suggest that LN storage is the best method of long-term storage of O. japonica spores. The critical water content of O. japonica spores is about 6% and reduction of the water content to this level improves outcome after LN storage greatly. The reason for various responses of O. japonica spores to desiccation and storage temperatures are discussed.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12–25°C

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.Abbreviations BAP 6-Benzylaminopurine - KIN 6-Furfurylaminopurine     

Germination in Zostera japonica is determined by cold stratification, tidal elevation and sediment type

The effects of water temperature and bottom sediment type were studied on seed dormancy and germination of Zostera japonica Ascherson & Graebner in mesocosm. To test whether the germination rate is affected by cold stratification, seeds were divided into two groups: those exposed to cold (7 °C) and those left untreated (23–15 °C). Additionally, to mimic tidal variation, we used five tidal depth treatments for germination experiments in mesocosm. In mesocosm tanks, there was a wide range of daily fluctuating temperature at datum line +40 cm (17–25 °C), D.L. +20 cm (15 °C), and D.L. +0 cm (4–7 °C). In contrast, the maximum temperature range at D.L. −20 cm and −40 cm was narrow (5–6 °C). In the no cold stratification group, the maximum germination rates on sandy, muddy sand, and muddy bottom sediment were 3%, 11%, and 3%, respectively. In the cold stratification group (7 °C), the maximum germination rates were 40%, 53%, and 54%, respectively. First germination was observed 36 ± 0 days and 43 ± 6 days after the start of the germination experiment in the cold stratification group and the no cold stratification group, respectively. Bottom sediment type and tidal level did not affect seed germination in the both stratification group. Cold stratification strongly increases germination in all sediment types tested and under varying temperature regimes and at different tidal levels. We also tested whether seed germination is affected by daily fluctuations in temperature (10 °C constant, 15 °C/10 °C, and 20 °C/10 °C were compared) in an indoor incubator. Forty-two days after being sown, the maximum seedling emergence rates in the three groups were 3 ± 5%, 21 ± 7%, and 42 ± 26%, respectively. At 20 °C/10 °C, first germination was observed 11 days after the start of incubation, the germination rate rose sharply after 18 day of incubation, and then it leveled off after 32–42 days of incubation. In the no cold stratification group, seed germination was not observed in any of the three treatments. This finding suggests that the breaking of seed dormancy and germination of Z. japonica seeds are determined strongly by cold temperature and daily fluctuations of temperature, respectively.     

Frozen storage stimulates the formation of embryo-like structures and elongating shoots in explants from mature Larix decidua and L. x eurolepsis trees

Branches were collected from a Larix decidua and a L. x eurolepis tree, both 36 years old, in mid-winter. These branches were placed in freezers at –5°C, –10°C, and –18°C. Primordial shoot explants were excised after 2 to 9 months of frozen storage. The material remained viable at all three temperatures for at least 9 months. The frozen storage stimulated formation of embryo-like structures that were capable of forming shoots with elongated stems.Abbreviations 1/2 LM half strength Litvay medium     

Optimized protocol for the pilot-scale preparation of fungal cellulase

Summary A 25-l scale protocol is devised for the optimal secretion and recovery of fungal cellulase. Using a selected higher yieldingTrichoderma viride SMC strain, a protocol consisted of: a) an optimized production medium rich in microcrystalline cellulose (MCC), fortified with 1% (w/v) ammonium sulphate, 0.5% (w/v) soybean flour, 0.1% (v/v) Tween-80 and other trace nutrients; b) optimized physical parameters of production, such as an inoculum containing a homogeneous suspension of 6×10⁷ conidia per 1,28±1°C, pH 4.0±0.5, 300±20 rpm, 11000±1000 l/h aeration, and 170–220 h duration; c) optimal recovery through a filter press (450 l/h rate of filtration) followed by precipitation with 2.5–3.0 volumes of acetone (15°C and basket centrifugation (27°C, 1700 rpm)); and d) vacuum drying (35°C, 4–6 h). This afforded 70% recovery of cellulase in the form of white fluffy powder containing 20000±2000 carboxy methyl cellulase and 1000±50 units filter paperase per g activities, with raw material cost of US$ 8–10 per million carboxy methyl cellulase units. During storage for 18 months at 4°C, ambient temperature and 37°C, the cellulase preparation was found to retain 100, 75 and 60% of its initial activity, respectively.     

Effects of temperature and solar radiation interactions on the survival of quiescent conidia of the entomopathogenic hyphomycetePaecilomyces fumosoroseus (Wize) Brown and Smith

The detrimental effect of solar radiation on the survival of conidia of the entomopathogenic fungusPaecilomyces fumoroseus was studied by monitoring germinability and ability to form colonies (CFU) of conidia irradiated at two temperatures, 25 and 35 °C, harmless to shaded conidia. There was no apparent effect when spores were exposed to a high level of artificial radiation (0.66 W m^–2 UVB). However, at a lower level of irradiance (0.33 W m^–2), effects of radiation occurred more quickly at 35 °C than at 25 °C. Under natural solar radiation, the rate of decrease in germinability or viability was doubled at 35 °C as compared to 25 °C, indicating an interaction between temperature and radiation effects under natural conditions. This interaction was not detected in indoor experiments, indicating that the spectral distribution of UV radiation has to be taken in account as well as its irradiance when studying its effects.Abbreviations CFU Colony Forming Units - UTC Universal Time Coordinates - UVB Ultra Violet B radiation (280–320 nm)     

The temperature-dependent duration of development and parasitism of three cereal aphid parasitoids, Aphidius ervi, A. rhopalosiphi, and Praon volucre

Temperature dependencies were established for the egg-to-mummy and mummy-to-adult phases, for mummy mortality, and for parasitism of Aphidius ervi Haliday, Aphidius rhopalosiphi De Stefani-Perez, and Praon volucre (Haliday) (Hymenoptera, Aphidiidae), three parasitoids of Sitobion avenae (Fabricius) (Homoptera, Aphididae), at 8°C, 12°C, 16°C, 20°C, and 25°C on winter wheat (cv. Haven). A physiological model described temperature-dependent development over the full temperature range, whereas a linear model was fitted for data above 8°C and used to estimate the lower temperature thresholds and day-degrees (° D) required for development. The thresholds for A. ervi were 2.2°C for egg-mummy development and 6.6°C for mummy-adult development, those for A. rhopalosiphi were 4.5°C and 7.2°C, and those for P. volucre were 3.8°C and 5.5°C. The time to develop into mummies and adults differed significantly between the three species: A. ervi development into mummies required an average of 159 ° D, while development into adults took an average of 73 ° D. The corresponding average times required for A. rhopalosiphi and P. volucre to develop mummies were 124° D and 126° D, while their development into adults required an average of 70° D and 150° D, respectively. Mummy mortality was 25–35% at 8°C and less at the higher temperatures tested, but began to increase again at 25°C, showing a quadratic relationship between mortality and temperature. Parasitization was very low or, in the case of P. volucre, absent up to 12°C and thereafter increased with increasing temperature. The relationship between parasitization, recorded as percent aphids mummified, and temperature was linear at the temperatures tested and depended on species. A. ervisuperparasitized 11.1% aphids at 20°C and 16.6% aphids at 25°C, whereas superparasitism was low in A. rhopalosiphi and absent in P. volucre. From 16°C to 25°C the P. volucre sex ratio increased. For A. ervi and A. rhopalosiphi there was no trend with temperature, but at 20°C and 25°C it was close to even. Field data for 1996 and 1997 allowed for a comparison of actual and expected emergence of overwintering mummies. In both years, parasitoids were predicted to have emerged from overwintering mummies well in advance of the onset of aphid infestation, and more than a month earlier than the first parasitized aphids were found in winter wheat. Observations from trap plants in other crops supported the predictions of the models. Other factors that can affect biological control by cereal aphid parasitoids are discussed.     

Heterosis in crosses between lines of Drosophila melanogaster selected for adaptation to different environments

Summary Experiments were designed to examine whether heterosis would occur in crosses of Drosophila melanogaster populations adapted to 18 °C or 28 °C environments. Crosses were examined in parental environments, an intermediate environment (23 °C) and a mixed environment (alternating 18°/28°C). Parental populations did not show divergence for larval viability, cold shock or high temperature mortalities when tested in a common environment. However, the 28 °C population was less fecund than the 18 °C population, but had higher larval competitive ability and higher adult longevity. Heterosis for viability, cold shock mortality and high temperature mortality occurred in crosses between a population adapted to 18 °C and another adapted to 28 °C, but not in crosses between two populations adapted to the same temperature. The results suggest that, in the absence of drift, heterosis is expected in crosses between lines or populations with different histories of selection but not between lines with the same selection histories.     

Supercooling capacity of Eurasian and North American populations of parasitoids of the Russian wheat aphid, >Diuraphis noxia

Supercooling points were estimated for seven populations of >Aphelinus albipodus, five populations of >Aphelinus asychis, and four populations of >Diaeretiella rapae to assess whether their supercooling points were sufficiently low to provide the potential for overwintering survival in colder temperate climatic areas. Test individuals from all 16 of the parasitoid populations were collected originally from mummies of the Russian wheat aphid, >Diuraphis noxia. Mummies containing parasitoid pupae were maintained for 1 wk under three different temperature conditions (treatments): at room temperature (24.8 ± 0.2 °C), 1 wk at 0 °C, and 1 wk –5 °C, and the supercooling points across treatments, and within and among species were compared. Statistical differences in supercooling points were found among populations of >A. albipodus for each treatment, and for >A. asychis when maintained for 1 wk at room temperature. No differences in supercooling points were found among populations of >D. rapae mummies maintained under the three temperature treatments. The lowest supercooling points obtained for the three parasitoid species maintained at room temperature were the >A. albipodus population from Montana (–31.68 °C), the >A. asychis population from Greece (–32.04 °C), and the >D. rapaepopulation from the Caucasus (–33.12 °C). Preconditioning the parasitoid mummies to cold had no effect on the supercooling points for >A. albipodus, and in some cases unexpectedly increased the supercooling points for >A. asychisand >D. rapae. In comparing the overall mean supercooling points of the three parasitoid species, no differences were found within species (among temperature treatments), nor among species (within temperature treatments). It was concluded that observed differences in supercooling points of only a few degrees Centigrade among parasitoid populations and species would not be expected to cause differences in their overwintering success, especially given the expected variability in temperatures within and among overwintering sites.     

Application of gas-permeable bags for in vitro cold storage of strawberry germplasm

Summary This study reports the first use of gaspermeable, heat-sealable polyethylene bags for cold storage of plant tissue cultures. The bags were used to develop a new cold storage system for the in vitro strawberry collection at the National Clonal Germplasm Repository (NCGR), Corvallis. In vitro Fragaria plantlets of 96 different accessions (species and cultivars) were transferred to bags with basal medium without growth regulators, heat-sealed, grown for one week at 25°C, cold hardened for one week, and then stored in the dark at 4°C. These in vitro cultures were successfully stored for up to 24 months in polyethylene bags. Evaluations at three month intervals provided information on the condition of the diverse collection. Over 75% of the accessions originally stored remained in storage for 15 months and 47% remained for over 18 months. None of the 96 accessions studied was lost due to contamination or decline in vigor. Over 300 Fragaria accessions are currently stored using this system.Abbreviations BA N⁶-benzyladenine - IAA indole-3-acetic acid - GA₃ gibberellic acid     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.