Conidia Of Metarhizium Anisopliae Research Articles

The Density and Viability of Metarhizium anisopliae Conidia on Several Growth Media

Metarhizium anisopliae is a parasitic fungus on insects, and thus called entomopathogenic fungus. This fungus is used as a biological control agent for insect pests. Fungal propagation can be done using a variety of media. The purpose of this study was to analyze the growth of M. anisopliae on four types of media, with conidial density and viability as the growth parameters. This research was conducted at the Laboratory of Microbiology, Universitas Negeri Semarang. This study was an experimental research used a Completely Randomized Design (CRD) with one factor and four treatments: Control (PDA/Potato Dextrose Agar medium), Treatment I (ELSA/Extract Larvae Sucrose Agar medium), Treatment II (CWSA/Coconut Water Sucrose Agar medium), Treatment III (CWELSA/Coconut Water and Extract Larvae Sucrose Agar medium). The and results showed that there was an effect of growth media on the density and viability of M. anisopliae conidia. CWELSA media had the highest conidial density (2.91 x 108 cfu/mL) and viability (97.17%). CWSA media had 2.82 x 108 cfu/mL and 95.33%. PDA media had 2.25 x 108 cfu/mL and 92.83%. ELSA media had 1.64 x 108 cfu/mL and 90.83%. The high conidial density and viability of M. anisopliae is CWELSA medium. This study is as an alternative growth medium to improve the quality of M. anisopliae propagation.

Prospects of fungal endophytes in the control of Liriomyza leafminer flies in common bean Phaseolus vulgaris under field conditions

Field trials were carried out for two seasons in two sites (Sagana and Naromoru, Central province of Kenya) to evaluate the prospects of endophyte isolates of Beauveria bassiana (Balsamo) Vuillemin G1LU3 and Hypocrea lixii Patouillard F3ST1 for the control of Liriomyza leafminer in common bean Phaseolus vulgaris L. crops through seeds inoculation. Autodissemination device (AD) treated with conidia of Metarhizium anisopliae (Metschnikoff) Sorokin ICIPE 20 was also added as a treatment. Leafminer infestation was not significantly different during the first season but was higher in the controls than in endophtyte treatments at both sites during the second season. Three key Liriomyza species [(L. huidobrensis Blanchard, L. sativae Blanchard and L. trifolii (Burgess)] and six parasitoid species [(Opius dissitus Muesebeck, Phaedrotoma scabriventris Nixon, Diglyphus isaea Walker, Neochrysocharis formosa Westwood, Hemiptarsenus varicornis Girault and Halticoptera arduine (Walker)] were identified during the trials. Leafminer infestation, number of pupae, leafminer flies and parasitoids emergence and yield were the parameters evaluated. Both isolates successfully colonized different parts of P. vulgaris plants, although the colonization was higher with H. lixii F3ST1 than B. bassiana G1LU3 at both sites. The mean number of pupae from the infested leaves varied between 141–252 and 331–416 in endophyte and control treatments, respectively, during the first season and from 110–223 to 366–523, respectively, in endophyte and control treatments during the second season. There were no significant differences among the treatments in the number of parasitoids that emerged from pupae. Higher yield of P. vulgaris seeds was obtained in endophyte than in control treatments. The inclusion of AD treatment did not have significant effect on all the parameters evaluated, except yield. Results of the present study suggest that both fungal isolates hold potential for pest management and could be considered for the control of leafminer flies. However, there is the need to confirm these results on large-scale trials.

Effectiveness of Metarhizium anisopliae formulations against dengue vectors under laboratory and field conditions

ABSTRACTThe oviposition behaviour of Aedes aegypti and the effectiveness of Metarhizium anisopliae conidia formulated in water or oil-in-water against A. aegypti adults and eggs were tested in multi-choice and no-choice tests in oviposition devices under laboratory conditions. Both females and males rested in the devices, regardless of the formulation, and were not repelled by the presence of conidia (up to 106 conidia/cm2) without oil or formulated with oil on treated filter paper arranged in the device. However, at higher oil concentrations (≥0.1 μl/cm2), regardless of the presence of conidia, the number of eggs laid by gravid females on the filter paper dropped. The susceptibility of adults, especially of males, to fungal infection increased up to a 15-day incubation. An elevated number of larvae (≥41%) eclosed from eggs laid on the moistened filter paper in the device even without submersion of eggs in water, and these larvae subsequently died. In the laboratory, 1 μl/cm2 oil combined with 106 conidia/cm2 clearly reduced eclosion to 1.8% after submersion of eggs in water compared to ≥13% eclosion in the control. In field tests in Goiânia, Brazil, eclosion of aedine larvae from eggs laid on filter paper previously treated with oil-in-water formulated conidia dropped to between 0% and 36% compared to 22–50% in the control. Promising results of laboratory and field tests with M. anisopliae formulated in water or oil-in-water and tested in a device emphasised the effectiveness of a fungus-based formulation for aedine mosquitoes in peridomestic areas.

Conidial surface proteins of Metarhizium anisopliae: Source of activities related with toxic effects, host penetration and pathogenesis

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

Method for determining the best hydrophilic-lipophilic balance (HLB) number for a compatible non-ionic surfactant in formulation development for aerial conidia of Metarhizium anisopliae (Hypocreales: Clavicipitaceae)

A direct dropping method was developed to determine the best hydrophilic-lipophilic balance number of Metarhizium anisopliae conidia at different water content levels during dehydration. Results showed the best HLB number of the tested M. anisopliae conidia was 8; dehydration did not change the best HLB number but significantly increased wetting time.

Evaluating physical and nutritional stress during mycelial growth as inducers of tolerance to heat and UV-B radiation in Metarhizium anisopliae conidia

Elevated tolerance to UV-B radiation and heat may be induced in conidia produced on fungi exposed during mycelial growth to sublethal stresses other than heat or UV-B. This is due to a phenomenon referred to as ‘cross-protection’. Several mechanisms are associated with this increased conidial tolerance, one of which is the accumulation of trehalose and mannitol within conidia. In the present study, conidia of the insect-pathogenic fungus Metarhizium anisopliae var. anisopliae were produced on mycelium subjected to nutritive, heat-shock, osmotic, or oxidative stress. The tolerance levels to UV-B radiation and heat of the conidia from stressed mycelium were evaluated, and the amounts of trehalose and mannitol accumulated in conidia were quantified. Conidia produced under nutritive stress (carbon and nitrogen starvation) were two-times more heat and UV-B tolerant than conidia produced under rich (non-stress) nutrient conditions [potato–dextrose agar with yeast extract (PDAY)], and they also accumulated the highest concentrations of trehalose and mannitol. Conidia produced on heat-shock stressed PDAY cultures had higher tolerance to UV-B radiation and heat than conidia produced without heat shock; however, both the UV-B tolerance and trehalose/mannitol concentrations in conidia produced on heat-shocked mycelium were less than those of conidia produced under nutritive stress. Conidia produced under osmotic stress (sodium or potassium chloride added to PDAY) had elevated heat and UV-B tolerances similar to those of conidia produced under nutritive stress; however, they had the lowest levels of mannitol and trehalose, which indicates that accumulation of these compounds is not the only mechanism used by M. anisopliae for protection from heat and UV-B radiation. Oxidative stress from UV-A irradiation or hydrogen peroxide did not produce conidia with elevated UV-B or heat tolerances. Conidia produced under oxidative stress generated by menadione had increased or unchanged tolerances to heat or UV-B, respectively. The levels of mannitol or trehalose in conidia were similar to those in the unstressed controls. Conidial yield was reduced, in some cases severely, by nutritive and osmotic stress; whereas oxidative and heat-shock stress did not alter levels of spore production.

A novel method for the quantitative assessment of the percolation of Metarhizium anisopliae conidia through horticultural growing media

Incorporation of fungal biological control agents (BCAs) into plant growing media has considerable ergonomic and economic benefits for growers. These agents usually give prophylactic control of target pests and diseases. However, their efficacy is dose dependent and loss of inoculum through leaching could influence the degree of protection they provide. At present there are no protocols to determine the loss of inoculum in the disparate growing media used in horticulture. We describe a method based on a nutrient leaching column to quantify leaching of conidia of the insect-pathogenic fungus Metarhizium anisopliae in a range of growing media. Conidia of this biocontrol agent were applied as a drench or premixed into the medium. Both the application method and growth medium influenced conidial leaching. Inoculum losses were greater following drench application than premixing (95% vs. 15%) irrespective of media type. Comparatively more inoculum was lost from bark and coir following drench application whereas losses were relatively high in peat following premixed application. The leaching column assay provided a simple and accurate method to quantify inoculum loss in real time. This assay could help determine leaching of other fungal BCAs in growing media. It could help in improving pest and disease control by optimizing the rate and frequency of conidial application as well in the design of more efficacious formulations.

Efficacy of strips coated with Metarhizium anisopliae for control of Varroa destructor (Acari: Varroidae) in honey bee colonies in Texas and Florida

Strips coated with conidia of Metarhizium anisopliae (Metschinkoff; Deuteromycetes: Hyphomycetes) to control the parasitic mite, Varroa destructor (Anderson and Trueman) in colonies of honey bees, Apis mellifera (Hymenoptera: Apidae) were compared against the miticide, tau-fluvalinate (Apistan) in field trials in Texas and Florida (USA). Apistan and the fungal treatments resulted in successful control of mite populations in both locations. At the end of the 42-day period of the experiment in Texas, the number of mites per bee was reduced by 69-fold in bee hives treated with Apistan and 25-fold in hives treated with the fungus; however mite infestations increased by 1.3-fold in the control bee hives. Similarly, the number of mites in sealed brood was 13-fold and 3.6-fold higher in the control bee hives than in those treated with Apistan and with the fungus, respectively. Like the miticide Apistan, the fungal treatments provided a significant reduction of mite populations at the end of the experimental period. The data from the broodless colonies treated with the fungus indicated that optimum mite control could be achieved when no brood is being produced, or when brood production is low, such as in the early spring or late fall. In established colonies in Florida, honey bee colony development did not increase under either Apistan or fungal treatments at the end of the experimental period, suggesting that other factors (queen health, food source, food availability) play some major role in the growth of bee colonies. Overall, microbial control of Varroa mites with fungal pathogens could be a useful component of an integrated pest management program for the honey bee industry.

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.

The germination of oil-formulated conidia of the insect pathogen, Metarhizium anisopliae

Conidia of Metarhizium anisopliae need water activity (Aw) > 0.98 for germination irrespective of whether they are formulated in an aqueous or oil carrier. The presence of nutrients in the carriers or culture medium accelerated germination but did not increase the range of Aw (or humidities) over which conidia could germinate. Insect extract stimulated more conidia to germinate than yeast extract. Oils appeared to extract fungistatic and stimulatory compounds from the insect cuticle. The ratio of these compounds depends on both the insect species and the carrier. Conidia of M. anisopliae formulated in oils were observed with SEM to flow over the surface of insect and plant cuticles. The conidia were probably deposited at sites which are conducive for germination and infection and consequently increased the overall mortality of insects. In contrast, aqueous formulations of conidia remain as drops on the leaf surface immediately after application with a pipetteman or sprayer.

Operational and Economic Analysis of a West African Pilotscale Production Plant for Aerial Conidia of Metarhizium spp. for Use as a Mycoinsecticide Against Locusts and Grasshoppers

Aerial conidia of Metarhizium anisopliae (flavoviride) var. acridum strain IMI 330189 used for the inundative biological control of grasshoppers and locusts in sub-Saharan Africa are produced in a purpose-built facility at the International Institute for Tropical Agriculture in Benin using a standard, two-stage mass-production system. The yields average 31.1 g of dry conidia powder/kg of rice substrate, the production capacity is 300-350 kg of conidia/year and the production costs are estimated at US$21/100 g (the recommended dose for 1 ha). The production process parameters vary within narrow limits established during optimization, but the yield is characterized by a high level of variation over time. The incubation period and temperature are identified as key factors, although they account for less than 40% of the yield variation. The variation in conidial viability and contamination are correlated with several parameters, but none can adequately explain this variation. The handling time, a principal limiting factor, could be reduced by increasing the substrate quantity/unit of production. An awareness of these factors presents the opportunity to fine tune production, although the options for increasing or improving production efficiency are limited within the constraints of the system.

Efficacy of Metarhizhium anisopliae for Control of Black Field Crickets, Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) in Pastures

The black field cricket, Teleogryllus commodus (Walker), is a serious pest of pastures in the winter rainfall zone of western Victoria. The most damaging stages in its life cycle are late instar nymphs and young adults, which occur in March and April. Current control is with a malathion/cereal bait applied each season from the air over large areas. In response to farmer demands for a biological alternative, the use of a mycoinsecticide with conidia of Metarhizium anisopliae as the active ingredient was investigated. Natural infections of M. anisopliae are widespread but rarely kill more than 5 % of the population. A number of isolates are available and all are genetically very similar. In 1995, a small field trial was established with FI-1099 applied at the rate of 2 or 4 x 10 3conidia/ha. The higher dose was the more effective and gave 60-70% control compared to over 80% control with the malathion check. Further laboratory screening identified a more virulent isolate, FI-1037, and this was used in a field trial in 1996. In this trial, an oil based ULV spray of FI-1037, applied at 1.5 x 10 13 conidia/ha, was as effective as the standard malathion treatment. This dose is probably still uneconomically high, and it is hoped that field trials using lower doses will be undertaken in the future.

Ultrastructural study of the penetration by Metarhizium anisopliae through Dimilin-affected cuticle of Manduca sexta

Fourth instar larvae of the tobacco hornworm, Manduca sexta, were inoculated with conidia of Metarhizium anisopliae then fed a diet containing the insect chitin synthesis inhibitor Dimilin. Cuticle of Dimilin-treated insects provided reduced resistance to penetration by hyphae of M. anisopliae. Widespread histolysis of postecdysial (Dimilin-affected) cuticle occurred. In addition, although lamellate preecdysial cuticle was not affected by Dimilin, the majority of the cuticle in the vertical cuticular columns was laid down at the same time as the postecdysial cuticle. Therefore, the vertical cuticular columns were areas of weakness in the preecdysial cuticle which as a consequence failed to provide a mechanical barrier to the penetrating fungus.