Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae

Metarhizium anisopliae conidia were formulated with three granular carriers and nine dust diluents and stored over an 8- to 12-month period at 4° or 20°C. The virulence of formulations, with the exception of two dust preparations, was reduced significantly compared to unformulated conidia against Culex pipiens pipiens larvae. The formulation components most detrimental to conidial virulence were corn cob granules, diatomaceous earth, and two Kaolinite diluents. This was exampled by a decline in virulence from ca. 100% for unformulated conidia to 36% or below for these formulations. LT₅₀ values also increased from 2.4–2.6 days for unformulated conidia to above 6 days. In contrast, a diluent derived from dried castor oil (Thixcin R) significantly enhanced conidial virulence at several doses above that of unformulated conidia against C. pipiens larvae. Enhancement occurred whether conidia were formulated prior to storage or stored separate from the diluent and mixed prior to application. The Thixcin R formulation was more effective against Anopheles stephensi larvae, but virulence was reduced against Aedes aegypti larvae. A bentonite formulation (Bentone-38) also maintained conidial virulence effectively, but Thixcin R was a superior diluent. It was shown that conidial virulence of formulations was not correlated with differences in conidial viability. The preparations that were applied dry by a surface method were more virulent than when an aqueous suspension containing a surfactant was used. The results demonstrate the need to assess efficacy of mycoinsecticidal formulations in a virulence bioassay prior to field testing.     

Effect of formulation on the viability of Metarhizium anisopliae conidia

A slide agglutination test using antibody-sensitized latex particles was developed for the specific detection in the early infection of the flacherie virus of the silkworm, Bombyx mori. With this test, 0.63 μg/ml of virus protein could be detected. The tests was completed within 5 min. Extracts from flacherie virus-infected silkworm larvae agglutinated latex particles specifically, while there was no agglutination by extracts of normal and nuclear polyhedrosis virus-infected silkworm larvae. The results showed that the sensitivity and simplicity of this technique for the detection of flacherie virus were greater than those of conventional serological techniques such as the immunofluorescence test and the immunodiffusion test.     

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.     

Studies on parasitic castration: Aminopeptidase activity levels and protein concentrations in Ilyanassa obsoleta (mollusca) parasitized by larval trematodes

Specimens of the estuarine prosobranch Ilyanassa obsoleta collected in the vicinity of Charleston, South Carolina, during June, July, and August 1981, were found to be parasitized by larvae of the digeneans Cercaria dipterocerca, Himasthla quissetensis, Lepocreadium setiferoides, Microbilharzia variglandis, Microphalloides nassicola, Stephanostomum tenue, and Zoogonus lasius. The aminopeptidase activity levels and the total protein concentrations of the hemocytes and sera of these infected snails were determined. It was ascertained that snails parasitized by larvae of L. setiferoides had significantly higher levels of hemocytic aminopeptidase activity and hemocytic total protein concentrations than noninfected snails. Furthermore, snails parasitized by L. setiferoides larvae had significantly higher hemocytic levels of aminopeptidase activity than snails parasitized by larvae of S. tenue, Z. lasius, M. nassicola, and H. quissetensis.     

Studies on parasitic castration: Occurrence of a gametogenesis-inhibiting factor in extract of Zoogonus lasius (Trematoda)

Testes of nonparasitized Ilyanassa obsoleta were maintained in vitro in a modification of the medium originally devised by Burch and Cuadros [Nature (London)206, 637–638 (1965)] in order to ascertain whether a saline extract of the daughter sporocysts of Zoogonus lasius would cause the inhibition of spermatogenesis. This parasite is known to cause parasitic castration of I. obsoleta in nature. It has been ascertained that after 96 hr of incubation with the parasite extract the incorporation of [³H]thymidine is inhibited in some spermatogonia. This represents the first experimental evidence that some molecule(s) of parasite origin may be responsible for chemical parasitic castration.     

Acid production by Metarhizium anisopliae: Effects on virulence against mosquitoes and on detection of in vitro amylase,protease, and lipase activity

The imperfect fungus Metarhizium anisopliae infects and kills larvae of many insect species, including the mosquito Culex pipiens. Mutants of M. anisopliae selected for enhanced production of amylase have been found to have simultaneously acquired hypervirulence against C. pipiens larvae. In the present work, wild-type and some mutant strains of M. anisopliae were found to excrete an acid or acids which alter the pH of fungal cultures below that permissible for amylase activity. The amylase-enhanced hypervirulence mutants did not excrete acid. Detection of protease and lipase activities was complicated by the acid excretion. When this was taken into account, mutants having altered lipase or protease production were found to have no alteration in virulence against mosquito larvae. The link between acid excretion, amylase activity, and hypervirulence is discussed.     

Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria.

The effects of relative humidity (RH) and temperature on the sporulation of Metarhizium anisopliae var. acridum on mycosed cadavers of desert locust, Schistocerca gregaria, were assessed in the laboratory. Quantitative assessments of conidial production over 10 days under constant conditions showed that sporulation was optimized at RH > 96% and at temperatures between 20 and 30 degrees C. Under both these conditions >10(9) conidia/cadaver were produced. At 25 degrees C, conidial yield was maximized under conditions in which cadavers remained in contact with damp substrate. Relatively little sporulation occurred at 15 degrees C (< 3 x 10(7) conidia/cadaver) and 40 degrees C (< 4 x 10(6) conidia/cadaver) and no sporulation occurred at 10 or 45 degrees C. Following incubation, conidial yield was closely related to the water content of locust cadavers. In separate tests, locust cadavers were incubated for 10 days under diurnally fluctuating temperature and RH that comprised favorable (25 degrees C/100% RH) alternating with unfavorable (40 degrees C/80% RH) conditions for sporulation. In this case, fewer conidia were produced compared with cadavers that were incubated under the favorable conditions for an equal period cumulatively but were not periodically exposed to unfavorable conditions. However, this reduced sporulation observed with the fluctuating condition was not observed when cadavers were similarly incubated under favorable/unfavorable conditions of temperature but were not periodically exposed to the low RH condition. This result implies that sporulation is a dynamic process, dependent not only on periodic exposure to favorable RH but also on the interrelation of this with low RH. Associated tests and the monitoring of changes in cadaver weights imply that the mechanism driving the reduced sporulation under fluctuating RH is the net water balance of cadavers, i.e. the cumulative ability of the fungus/cadaver to adsorb water necessary for sporulation at high RH is restricted by water loss associated with intermittent exposure to a low RH. The duration of daily exposure to high humidity appears to be a crucial constraint to the recycling ability of M. anisopliae var. acridum.     

Influence of whey permeate and millet as substrates on thermotolerance of Beauveria bassiana and Metarhizium anisopliae conidia during storage

Conidia from Beauveria bassiana and Metarhizium anisopliae, produced in quarter-strength Sabouraud dextrose agar amended with yeast extract, whey permeate, and millet agar, were exposed to 45°C for 90 min prior to and at 30 d post-storage at 25°C. B. bassiana produced in whey permeate or millet had better thermotolerance than the other treatments after the storage.     

Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera: Noctuidae)

The effect of repeated conidial sub-culturing of Metarhizium anisopliae on its virulence against Helicoverpa armigera (Hübner) was studied. The LT₅₀ observed against third instar larvae of H. armigera for the first sub-culture was 3.4 days; it increased to 4.5 and 5.6 days for the 20th and the 40th sub-cultures, respectively. The LT₅₀ values after passage of the 40th sub-culture on H. armigera decreased to 4.4 and 3.7 days for the 40th (first in vivo) and the 40th (fifth in vivo) passages, respectively. Similarly, the LC₅₀ of M. anisopliae towards third instar larvae of H. armigera increased from the first sub-culture (0.17×10⁴) to (3.0×10⁴) for the 40th conidial transfers on potato dextrose agar and again decreased to 0.74×10⁴ and 0.23×10⁴ in the 40th (first in vivo) and the 40th (fifth in vivo) passage, respectively. Similar trends for LC₅₀ and LT₅₀ values were seen when sugarcane woolly aphid, Ceratovacuna lanigera Zehntner was used as a host. Significant variation in appressorium formation and cuticle-degrading enzyme production such as chitinase, chitin deacetylase, chitosanase and protease during subsequent sub-culturing and passage through H. armigera was observed. Though there was no effect on internal transcribed spacer (ITS) sequence pattern, interestingly, in randomly amplified polymorphic DNA (RAPD), significant differences in the band intensities and in the banding pattern for different sub-cultures of M. anisopliae were observed. As stable virulence towards the insect pest is desirable for commercialisation of a mycoinsecticide, such changes in virulence due to repeated in vitro transfer need to be monitored and minimised.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Effects of conidial densities and spray volume of Metarhizium anisopliae and Beauveria bassiana fungal suspensions on conidial viability,droplet size and deposition coverage in bioassay using a novel bioassay spray system

A newly developed spray tower was used to characterise droplet distribution and coverage of conidial suspensions of Metarhizium anisopliae ATCC 62176 and Beauveria bassiana NI8 with different spray volumes. ATCC 62176 and NI8 had different spray models which could be caused by the surface physicochemical characteristics of the strains and conidia.     

Effects of virulence,sporulation, and temperature on Metarhizium anisopliae and Beauveria bassiana laboratory transmission in Coptotermes formosanus

Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.     

Predicting the effect of relative humidity on skin temperature and skin wettedness

The purpose of this research is to investigate the relative humidity effects on skin temperature and skin wettedness for different operative temperatures. For this aim, thermal interactions between human body and environment are simulated. In this simulation, Gagge 2-node model is used but includes some significant modifications. The simulation is to apply the Gagge 2-node model to individual segments rather than to whole body. Also, the results of the simulation are compared with present measurements, and available experimental data and simulated results in the literature in order to present reliability of the 16 segments-Gagge 2-node model. It is determined that the simulation results are in good agreement with measured results obtained from present experiments, and simulated results and experimental data in the literature.     

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×10¹² viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.     

温湿度对玫烟色棒束孢IF‐1106菌株孢子萌发及对烟粉虱致病力的影响(英文)

B型烟粉虱Bemisia tabaci Gennadius是一种重要的世界性农业害虫。玫烟色棒束孢Isaria fumosorosea是防治烟粉虱的一种重要的虫生真菌。对不同温度(20、23、26、29、32℃)、不同湿度(53%、65%、75%、85%、95%)下玫烟色棒束孢IF‐1106菌株分生孢子萌发及其对烟粉虱的致病力进行了测定。结果表明:温度对孢子的萌发有显著影响,26℃时孢子萌发率最高。当相对湿度低于75%时,孢子不萌发或萌发率较低;当相对湿度为85%–95%时,孢子萌发率显著升高。26℃时,烟粉虱2龄若虫的累计死亡率最高。随着相对湿度增大,病菌的致病力增强。当相对湿度为53%–95%时,烟粉虱2龄若虫累计死亡率从54.55%增加到88.89%。玫烟色棒束孢的致病力与孢子萌发率呈正相关,但温度比相对湿度对其影响更明显。结果表明,玫烟色棒束孢IF‐1106菌株侵染烟粉虱的最佳条件是温度26℃和相对湿度大于85%。     

Studies on the regeneration-restore and karyotype of protoplast in Metarhizium anisopliae var. majus

Abstract:  The characteristics and regeneration-restore of protoplasts and its karyotype of an insect pathological fungus, Metarhizium anisopliae var. majus were studied. Among the protoplasts, 25.3% were without a nucleus, and 74.7% contained a nucleus. Among the nucleus protoplasts, 53.6% contained a single nucleus. The regeneration-restore of protoplasts was of three distinct shapes. Considering the frequency of regeneration and the growing speed of the colony, 0.7 mol/l glucose was the optimum as osmotic stabilizer of culture medium in the regeneration-restore of the protoplasts. The chromosomal DNA molecules of M. anisopliae var. majus have been separated into seven bands by pulsed-field gel electrophoreses. Using the Schizosaccharomyces pombe chromosomes as size standard, the size of chromosomal DNA was estimated to be 1.1–6.5 Mb and its karyotype exhibited polytypism among strains.     

Impact of temperature and relative humidity on effectiveness of Metarhizium guizhouense PSUM02 against longkong bark eating caterpillar Cossus chloratus Swinhoe under laboratory and field conditions

The longkong bark eating caterpillar, Cossus chloratus Swinhoe (Cossidae: Lepidoptera), is a serious pest of longkong trees Lansium domesticum Corrêa that affects quality and quantity of the fruit. For eco-friendly management of this pest, Metarhizium guizhouense PSUM02, an entomopathogenic fungus, was tested under laboratory and field conditions of Thailand. Relative humidity (RH) more than 93% provides the highest virulence to M. guizhouense PSUM02 by reducing the survival of C. chloratus larvae to 0.0–18.0%. The average survival time (AST) of C. chloratus larvae infected with M. guizhouense PSUM02 at 100% RH showed the lowest AST of 3.9 ± 0.2 days. The optimum temperature for M. guizhouense PSUM02 to infect C. chloratus larvae was 25–30 °C with 2–20% survival after 10 days of treatment. The AST of C. chloratus larvae infected with M. guizhouense PSUM02 at 25 °C or 30 °C was 5.8 ± 0.6 or 3.4 ± 0.2 days, respectively, which was significantly lower than the AST observed at 20 °C and 35 °C temperatures. M. guizhouense PSUM02 showed effectiveness under field conditions by reducing the number of larvae of longkong bark eating caterpillar C. chloratus in treated longkong tree. The application of M. guizhouense PSUM02 could be an effective option for the eco-friendly management of longkong bark eating caterpillars in the longkong orchards of Thailand and other longkong producing countries.     

Synergistic effect of relative humidity and temperature on the survival of rhizobia in inoculant carrier

Six strains of Rhizobium , three temperature-tolerant (U1, C13 and A19) and three temperature-sensitive strains (U10, C10 and A4) selected on the basis of previous study were used to screen the synergestic effect of different relative humidities (r.h. 50%, 65% and 90%) and temperatures (28°, 35°, 40° and 45°C) on the survival of rhizobia in inoculant carrier. At a particular temperature all the three r.h.'s were maintained. At a storage temperature of 28° and 35°C, the r.h. had little effect on the population of any of the rhizobial strains tested, but at 40° and 45°C, marked differences were observed and it was concluded that higher r.h. in conjunction with higher temperature resulted in low viable counts. The effect was similar but less obvious with the temperature-tolerant strains. It was found that 50% r.h. at different storage temperatures extended shelf life of rhizobial strains of blackgram, cowpea and arhar crops which were tested in this study in inoculant carrier.