Effects of Moisture Content and Temperature on Storage of Metarhizium flavoviride Conidia

The effects of moisture content and temperature on the medium-term (3-4 months) storage of conidia of Metarhizium flavoviride were investigated. Conidia harvested after 24 days of culturing on rice showed greater tolerance to long storage than conidia from 12-day cultures. The moisture content of the conidia was of greatest importance; at harvest from the culture, conidial moisture contents could be 40%, while the optimal moisture content for storage was found to be 4-5%. Dried conidia stored in oil benefited from the addition of dried silica gel, as did conidia stored as powder. A range of mineral oils proved satisfactory for storage, and when dried silica gel was added to suspensions, germination levels were 79.8% after 105 days at 28-32 C. Dried conidia stored in oil maintained germination levels of up to 96 and 85% after 80 days at 10-14 C and 28-32 C respectively. Dried conidia stored as powder retained germination levels of 95% at 10-14 C, but only up to 27% at 28-32 C. In another experiment, dried conidia maintained greater than 90% germination over 128 days, with or without silica gel at 10 - 14 C or -15 - -18 C.     

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.     

Seed germination and seedling growth in the arrow bamboo <Emphasis Type="Italic">Fargesia qinlingensis</Emphasis>

Improving natural regeneration of bamboos after they die following mass flowering is critical for conservation of giant pandas. However, little is known about factors that affect seed germination and seedling growth of bamboos. We studied seed germination and seedling growth in Fargesia qinlingensis, which mass flowered in a giant panda habitat in the Qinling Mountains of China in early 2000, in laboratory and greenhouse conditions. Seed germination rate was tested under light and dark conditions 5 and 12 months after seed collection. Germination rate displayed no significant difference under light or dark conditions 5 months after seed collection, but was significantly greater in the dark than under light 12 months after seed collection, suggesting light inhibition of seed germination. A 2×2 factorial design was conducted to test the effects of nitrogen (N fertilization and non-N fertilization) and light [full sun and shade (i.e., 14% full sun)] on seedling growth and biomass allocation. N fertilization significantly increased seedling growth, resulting in greater seedling height, more branches, more leaves, greater stem biomass, and greater leaf biomass. Seedlings under 14% full sun conditions had a significantly lower percentage of biomass allocated to the stem. The root/shoot ratio was significantly greater in non-N/shade than non-N/full sun, while there was no significant difference in this ratio between N/shade and N/full sun, suggesting that nitrogen fertilization compensated for the effect of shade on biomass allocation. Our results suggest that N fertilization could be employed in restoration of F. qinlingensis stands after die-off following mass flowering.     

Chemical treatment of soil to prevent transmission of tobacco rattle virus to potatoes by Trichodorus spp

Alternaria longipes (Ell. &Ev.) Mason survived on autoclaved maize stems for 6 months without losing its pathogenicity, but rapidly lost viability on non-autoclaved stems and could not be re-isolated 4 months after inoculation. In laboratory tests it infected both living and dead maize leaves. Some Alternaria isolates from non-solanaceous hosts infected tobacco leaves kept at high humidities for 10 days after inoculation, but not when this incubation period was reduced to 48 h. In the field, perennation on plants other than tobacco is unlikely to be important as a source of inoculum. Pathogenicity of Alternaria isolates was maintained from one season to the next when stored as conidia in sterile soil, or as dried, infected tobacco leaves; some isolates maintained on agar slopes under oil were still pathogenic after 5 years. Alternaria conidia collected from the surface of tobacco seedlings, and isolates from apparently healthy seedling leaves were pathogenic to mature tobacco. In the field conidia were detected on tobacco leaves soon after these emerged, and epiphytic colonies were occasionally found well in advance of symptoms. Many latent infections were also detected up to 5 weeks in advance of symptoms. Visual development of latent infections closely coincided with the end of leaf expansion.     

The effects of some storage conditions on viability of Lecanicillium lecanii conidia to whitefly (Homoptera: Trialeurodes vaporariorum)

A study on the survival of Lecanicillium lecanii conidia in storage at room temperature was carried out. Firstly, drying methods of conidia powder were compared. Vacuum-freeze drying (VFD) was more suitable for drying conidia as compared to vacuum drying (VD) at room temperature. Vacuum-freeze drying for 24-h resulted in a water content of 5.4%, and a viability, determined as germination of conidia in 2% glucose solution after16 h, was 90.3% and the infection in greenhouse whitefly, Trialeurodes vaporariorum was about 94.7% at a dose of 1×10⁸ conidia/mL. Secondly, the factors influencing viability of conidia stored at room temperature were evaluated in the laboratory. Temperature was the most critical factor influencing conidial storage stability, among the tested factors affecting survival of conidia stored at room temperature for 6 months. Both conidial germination and infection of hosts decreased with storage temperature increasing from 15 to 35°C, and at 35°C the survival of stored conidia for 6 months was near zero. The moisture content of the conidial powder was another major factor influencing viability of stored conidia at room temperature. Conidial powder dried to about 5% moisture content showed higher viability than non-dried conidial powder. For the carriers, clay and charcoal were more suitable for storage of L. lecanii conidia at room temperature. At a room temperature of 25°C, L. lecanii conidia which were dried to 5% water content and mixed with clay or charcoal could retain about 50% survival after 6 months' storage.     

Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.     

Some factors affecting germination of Peronospora farinosa conidia in water

The germination of conidia of Peronospora farinosa f. sp. betae, collected from sugar beet and suspended in deionized water, was inhibited by dilution with 10% solutions of glycerol, glucose or sucrose and with sap from sugar-beet leaves. Germination was stimulated by diluting with deionized water but not with tap water or biological saline. Substances that diffused from excised buds of sugar-beet plants into deionized water also stimulated germination of conidia but diffusates from leaves did not. This may partly explain why buds are more susceptible to downy mildew than leaves in sugar beet. Germination of conidia was apparently stimulated more by diffusates from buds of seedlings than by those from buds of older plants; this may help to explain why sugar-beet seedlings are more susceptible to downy mildew than older plants. Diffusates from plants of four sugar-beet stocks, that differed from each other in susceptibility to downy mildew, had very similar effects on germination of P. farinosa conidia. Stimulation of spore germination on the surfaces of buds and leaves did not seem, therefore, to be an important factor in determining resistance or susceptibility to downy mildew in these stocks.     

Post-harvest age-induced seed dormancy of Acer ginnala and its alleviation by growth regulator and low temperature treatments

One-month-old fruits of Acer ginnala with winged pericarp attached gave 44% germination and this was not increased by cold treatment at 4°C for 0, 10, 20, or 30 days, gibberellic acid treatment at 0, 1, 10, 100 or 1000 mg litre^-1, or ethephon treatment at 0, 2, 20, 200 or 2000 mg litre^-1. After 6 months of storage at 20–25 °C, germination of untreated fruits fell to 5% but could be restored to that of 1-month-old fruits by incubation at 4 °C for 30 days. After 9 months storage, no germination occurred in untreated fruits. Cold treatment (30 days at 4 °C partially restored germination (26%). Treatment with either gibberellic acid (1000 mg litre^-1) and 30 days at 4 °C (40%) or ethephon (100 mg litre^-] and 30 days at 4 °C improved germination (69%). The combination of all three treatments, i.e. 100 mg litre^-1 gibberellic acid, 100 mg litre^-1 ethephon and 30 days at 4 °C, optimised germination (86%). Thus, dormancy of A. ginnala developed during storage but could be reversed by a combination of treatment with low temperature and growth regulators. The highest germination (86%) was achieved after low temperature and growth regulator treatment of stored fruit.     

Long-term Storage of Metarhizium flavoviride Conidia in Oil Formulations for the Control of Locusts and Grasshoppers

Freshly harvested conidia of Metarhizium flavoviride (Gams & Rozsypal) were stored in two vegetable oils, groundnut or soya, or a mineral oil, Edelex. They were diluted with either Shellsol K or deodorized kerosene, and antioxidants were added to half of the vegetable oil formulations. Dried non-indicating silica gel was added to half of the formulations before storage at 8 or 17 C. Undried conidia, those without silica gel, lost viability rapidly, with germination dropping below 40% after 9 and 32 weeks at 17 and 8 C respectively. After 127 weeks (ca. 30 months) in storage, germination remained at over 60 and 80% for the dried formulations at 17 and 8 C respectively (after an unexplained drop in germination after 16-18 months in storage). Comparable figures for 160 weeks (ca. 37 months) were 47 and 68%. These figures represented germination after 24 h of incubation; after 48 h of incubation, germination was 79 and 89% from samples stored for 160 weeks at 17 and 8 C respectively. Representative formulations of the stored conidia were tested in bioassays against the desert locust Schistocerca gregaria (Forskal) up to 30 months into the experiment, and were found to have retained full virulence compared with freshly prepared formulations.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Survival of Conidia of Clonostachys rosea on Stored Barley Seeds and Their Biocontrol Efficacy Against Seed-borne Bipolaris sorokiniana

Survival and biocontrol activity of Clonostachys rosea (isolate IK726) conidia during storage on barley seeds were investigated. The initial density of colony forming conidia on seed was 4 &#50 10 3 to 9 &#50 10 4 colony forming units (cfu)/seed. After 5 months storage at 4&#176;C, the density decreased by less than one order of magnitude and the biocontrol efficacy against seedling blight caused by seed-borne Bipolaris sorokiniana was maintained at a significantly high level ( > 80% disease reduction) for > 5 months. Conidial survival on seeds stored at 20&#176;C declined more rapidly than at 4&#176;C, and biocontrol efficacy was significantly reduced after 3-5 months. However, conidia produced on solid media over 20 days survived better than conidia produced in liquid culture and conidia from solid media produced over 12 days. In contrast, when seeds treated with conidia were packed with silica gel and stored at 20&#176;C, the cfu density decreased by less than one order of magnitude after 5 months and the biocontrol efficacy was still high after 6 months. A dose-response curve revealed that 103 cfu/seed were needed for 80% control of seedling blight. Similar control was obtained in storage experiments when approximately 103 cfu/seed were recovered from seed, indicating that conidia which survived also retained a high ability to control disease.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.     

锰胁迫对盐肤木种子萌发、幼苗生长及理化特性的影响

为揭示植物适应锰胁迫的生理机制,通过在不同Mn²⁺浓度(0、1、5、10、15、20 mmol/L)下开展盐肤木(Rhus chinensis)种子萌发以及幼苗生长实验,检测锰胁迫处理7、15、30 d后幼苗生理生化特性的变化。结果表明:(1)随着Mn²⁺浓度的升高,盐肤木种子发芽率变化不显著,在80.0%-81.6%之间,发芽势、发芽指数和活力指数则呈先升后降的趋势;其幼苗生物量也呈现先升后降的趋势;(2)随着Mn²⁺浓度的升高与胁迫时间的延长,盐肤木幼苗叶绿素a、叶绿素b含量均呈现先增加后降低的趋势,类胡萝卜素含量呈现下降的趋势;(3)胁迫7 d时,随着Mn²⁺浓度的升高,盐肤木幼苗超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性均显著上升;胁迫15、30 d时,高Mn²⁺浓度(15-20 mmol/L)下POD、CAT活性则均降低;(4)胁迫7 d时,随着Mn²⁺浓度的升高,可溶性糖、可溶性蛋白、游离脯氨酸含量升高;胁迫15、30 d时,在Mn²⁺浓度为20 mmol/L时可溶性蛋白与游离脯氨酸含量显著降低;(5)随着Mn²⁺浓度的升高与胁迫时间的延长,丙二醛(MDA)含量均升高。研究说明盐肤木具有较强的耐受锰胁迫能力,它可通过增强抗氧化酶活性、积累渗透调节物质含量来应对锰胁迫。     

Ultra-violet radiation damage to Metarhizium flavoviride conidia and the protection given by vegetable and mineral oils and chemical sunscreens

Metarhizium flavouiride conidia formulated in oil or water were exposed to simulated solar radiation. Radiation below 320 nm killed conidia and caused delays in the germination of survivors; germination was greater after 48 h of incubation than after 24 h. UV exposure of conidia formulated in oil for 2 h reduced germination from 99% to 37.5% after incubation for 48 h. Exposure of conidia in water to UV for 1 h resulted in 4.7% germination after 24 h incubation compared with 36.5% for conidia formulated in oil. The addition of 1% oxybenzone resulted in 81.9% conidial germination after 3 h exposure and 48 h incubation compared with 28.1% in oil without the sunscreen.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

Biological fitness of Trichoderma atroviride during long-term storage,after production in different culture conditions

Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C?:?N 5?:?1 and C?:?N 160?:?1, indicating that C?:?N ratio in culture medium is not important for conidium survival of T. atroviride.