Virulence Of Metarhizium Anisopliae Research Articles

Evaluation of host–pathogen interactions for selection of entomopathogenic fungal isolates against Oligonychus afrasiaticus (McGregor)

Understanding the interaction between pathogen virulence and host responses is very important for selecting virulent isolates of entomopathogenic fungi that control an economically important pest, such as the date palm dust mite, Oligonychus afrasiaticus (McGregor). In this study, the virulence of Metarhizium anisopliae (ARSEF 8453 and ARSEF 9374), Beauveria bassiana (ARSEF 6884 and ARSEF 8037), and Isaria fumosorosea (ARSEF 9602 and ARSEF 7284) was evaluated by virulence-regulating traits and host detoxification-related enzymatic analysis. The results revealed significant differences in virulence among all tested isolates of entomopathogenic fungi. The most virulent isolate of B. bassiana ARSEF 6884 (LT50 = 6.3 days; LC50 = 2.7 × 106 conidia ml−1) observed in the current study revealed the highest activity of cuticle-degrading protease Pr1 (2.12 ± 0.10 μmol NA ml−1 min−1) compared with the least virulent isolate of I. fumosorosea ARSEF 7284 (LT50 = 13.1 days; LC50 = 7.9 × 107 conidia ml−1). Virulent isolates successfully infected the target host and revealed lower activities of antioxidant (catalase and superoxide dismutase) and detoxification (glutathione S-transferases) enzymes at late stage infection. In conclusion, isolates ARSEF 6884 and ARSEF 9374 were found to be promising for the development of an environmentally friendly management strategy to control date palm dust mites.

Virulence of Metarhizium anisopliae and Beauveria bassiana isolates and the effects of fungal infection on the reproduction potential of Rhiphicephalus microplus engorged females

ABSTRACTThe aim of the study was to assess the virulence of five Metarhizium anisopliae (Ma) and three of Beauveria bassiana (Bb) isolates, and the effect of the fungal infection to the reproduction of engorged females from two colonies of Rhiphicephalus microplus; one colony was collected from naturally infested cattle (Native) and the other one from a laboratory colony (Media Joya). Virulence was evaluated using the immersion technique at a concentration of 1 × 108 conidia/ml; control groups received a water suspension with Tween 80 (0.1%). The Reproductive Efficiency Index ‘REI’ (eggs laid/engorged female weight) and the Reproductive Aptitude Index ‘RAI’ (eggs hatched as larvae/engorged female weight) were calculated for both groups. This experiment shows that two entomopathogenic fungal isolates, Bb115 and Ma136, caused high mortality from 5 days post-treatment (PT), reaching mortality rates of 99–100% at 15 days PT in both R. microplus colonies. The Bb115 isolate caused 98 and 79% reduction in egg oviposition in the field and laboratory colonies, respectively, while the reduction in egg hatchability was 98 and 89% in the field and laboratories colonies, respectively. In the case of Ma136, the egg oviposition was reduced in 73% in the field colony and 64% in laboratory colony, while in the field and laboratory colonies, with a reduction in egg hatchability of 73% and 86%, respectively. These results indicate the potential of Bb115 and Ma136 isolates as possible biological control agents of R. microplus.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

INFECTION PROCESS OF ENTOMOPATHOGENIC FUNGI Metarhizium anisopliae IN THE Tetranychus kanzawai (KISHIDA) (TETRANYCHIDAE: ACARINA)

The importance of fungal attack to acarina has been observed but not yet on Tetranychus kanzawai . When infected mite, either within in a same species or even in a different species of entomopathigenic probably have similar signs and simptoms to mites. Characteristic of entomopathogenic fungus to Tetranychus kanza-wai was investigated. Three selected isolates of Metarhizium anisopliae , from Philippines and Indonesia were evaluated. The following aspects were investigated: (1) Bioassay tes of virulence, (2) Koch Postulate and (3) Infection process on each fungus against mite. Virulence of Metarhizium anisopliae was 1.4 X 10 3 , 7.2 X 10 2 , 5 X 10 2 PIB per ml on Ma4, Ma5 and Ma6, respectively. On Koch Postulates, Reinoculation and reisolation of all seven isolates from infected T. kanzawai showed definite signs of infection. This demonstrated its entomopathogenic capacity. In this experiment, adult mites were sprayed with 10 8 per ml concentrations of conidia observed under light microscope and Scanning Electron Microscopy (SEM). The result found T. kanzawai was very susceptible to three isolates M . anisopliae . In the end of the trials, fungal growth was detectable as early as 2 until 4 days of observation. Infection process with microphoto-graph and Scanning Electro Microscopy showed attachment, germination and penetration, extrusion and conidiogenesis fungal form. Keywords: Infection, Tetranychus kanzawai , Metarhizium anisopliae , Light micros-copy, Scanning Electro Microscopy (SEM) Normal 0 false false false EN-US X-NONE X-NONE Normal 0 false false false EN-US X-NONE X-NONE

INFECTION PROCESS OF ENTOMOPATHOGENIC FUNGI Metarhizium anisopliae IN THE Tetranychus kanzawai (KISHIDA) (TETRANYCHIDAE: ACARINA)

The importance of fungal attack to acarina has been observed but not yet on Tetranychus kanzawai . When infected mite, either within in a same species or even in a different species of entomopathigenic probably have similar signs and simptoms to mites. Characteristic of entomopathogenic fungus to Tetranychus kanza-wai was investigated. Three selected isolates of Metarhizium anisopliae , from Philippines and Indonesia were evaluated. The following aspects were investigated: (1) Bioassay tes of virulence, (2) Koch Postulate and (3) Infection process on each fungus against mite. Virulence of Metarhizium anisopliae was 1.4 X 10 3 , 7.2 X 10 2 , 5 X 10 2 PIB per ml on Ma4, Ma5 and Ma6, respectively. On Koch Postulates, Reinoculation and reisolation of all seven isolates from infected T. kanzawai showed definite signs of infection. This demonstrated its entomopathogenic capacity. In this experiment, adult mites were sprayed with 10 8 per ml concentrations of conidia observed under light microscope and Scanning Electron Microscopy (SEM). The result found T. kanzawai was very susceptible to three isolates M . anisopliae . In the end of the trials, fungal growth was detectable as early as 2 until 4 days of observation. Infection process with microphoto-graph and Scanning Electro Microscopy showed attachment, germination and penetration, extrusion and conidiogenesis fungal form. Keywords: Infection, Tetranychus kanzawai , Metarhizium anisopliae , Light micros-copy, Scanning Electro Microscopy (SEM) Normal 0 false false false EN-US X-NONE X-NONE Normal 0 false false false EN-US X-NONE X-NONE

English

The importance of fungal attack to acarina has been observed but not yet on Tetranychus kanzawai . When infected mite, either within in a same species or even in a different species of entomopathigenic probably have similar signs and simptoms to mites. Characteristic of entomopathogenic fungus to Tetranychus kanza-wai was investigated. Three selected isolates of Metarhizium anisopliae , from Philippines and Indonesia were evaluated. The following aspects were investigated: (1) Bioassay tes of virulence, (2) Koch Postulate and (3) Infection process on each fungus against mite. Virulence of Metarhizium anisopliae was 1.4 X 10 3 , 7.2 X 10 2 , 5 X 10 2 PIB per ml on Ma4, Ma5 and Ma6, respectively. On Koch Postulates, Reinoculation and reisolation of all seven isolates from infected T. kanzawai showed definite signs of infection. This demonstrated its entomopathogenic capacity. In this experiment, adult mites were sprayed with 10 8 per ml concentrations of conidia observed under light microscope and Scanning Electron Microscopy (SEM). The result found T. kanzawai was very susceptible to three isolates M . anisopliae . In the end of the trials, fungal growth was detectable as early as 2 until 4 days of observation. Infection process with microphoto-graph and Scanning Electro Microscopy showed attachment, germination and penetration, extrusion and conidiogenesis fungal form. Keywords: Infection, Tetranychus kanzawai , Metarhizium anisopliae , Light micros-copy, Scanning Electro Microscopy (SEM) Normal 0 false false false EN-US X-NONE X-NONE Normal 0 false false false EN-US X-NONE X-NONE

Influence of commercial fungicides on the germination, growth and virulence of four species of entomopathogenic fungi

The influence of 15 commercially available fungicides on the germination, growth and virulence of Metarhizium anisopliae, Beauveria bassiana, Isaria fumosorosea, and Lecanillium longisporum was evaluated. The influence of the fungicides on conidial germination was dependant on the fungicide type and dose. Most fungicides retarded conidial germination of all the fungi tested at 10× and at the recommended rate of application, however, their toxicity declined at lower concentrations. Most of the fungicides inhibited mycelial growth of B. bassiana, whereas L. longisporum growth was unaffected. Only two and eight fungicides influenced mycelial growth of I. fumosorosea and M. anisopliae, respectively. None of the fungicides influenced the virulence of B. bassiana and L. longisporum, however, tolylfluanid and azoxystrobin reduced the virulence of M. anisopliae and I. fumosorosea, respectively. These studies clearly show that certain fungicides have the potential to inhibit germination of entomopathogenic fungi in vitro but appear to have little or no effect on their virulence against target insects.

Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus

Growth under stress may influence pathogen virulence and other phenotypic traits. Conidia of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae (isolate ARSEF 2575) were produced under different stress conditions and then examined for influences on in vitro conidial germination speed, adhesion to the insect cuticle, and virulence to an insect host, Tenebrio molitor. Conidia were produced under non-stress conditions [on potato–dextrose agar plus 1 g l −1 yeast extract (PDAY; control)], or under the following stress conditions: osmotic (PDAY + sodium chloride or potassium chloride, 0.6 or 0.8 m); oxidative [(PDAY + hydrogen peroxide, 5 m m) or UV-A (irradiation of mycelium on PDAY)]; heat shock (heat treatment of mycelium on PDAY at 45 °C, 40 min); and nutritive [minimal medium (MM) with no carbon source, or on MM plus 3 g l −1 lactose (MML)]. Conidia were most virulent (based on mortality at 3 d) and had the fastest germination rates when produced on MML, followed by MM. In addition, conidial adhesion to host cuticle was greatest when the conidia were produced on MML. Media with high osmolarity (0.8 m) produced conidia with slightly elevated virulence and faster germination rates than conidia produced on the control medium (PDAY), but this trend did not hold for media with the lower osmolarity, (0.6 m). Conidia produced from mycelium irradiated with UV-A while growing on PDAY had somewhat elevated virulence levels similar to that of conidia produced on MM, but their germination rate was not increased. Hydrogen peroxide and heat shock treatments did not alter virulence. These results demonstrate that the germination, adhesion and virulence of M. anisopliae conidia can be strongly influenced by culture conditions (including stresses) during production of the conidia.

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.

Repeatedin vitrosubculturing alters spore surface properties and virulence ofMetarhizium anisopliae

Repeated subculturing caused rapid changes in the spore surface properties and virulence of Metarhizium anisopliae. Of the two strains evaluated, M. anisopliae V245 attenuated more rapidly than V275. Electrophoretic mobility and Radial Flow Chamber assays were used for the first time to generate qualitative and quantitative information on the adhesive forces of M. anisopliae conidia. Independent of strain, adhesion, hydrophobicity and spore-bound Pr1 declined after the first subculture; however, spore surface charge decline was erratic. Adhesion and hydrophobicity stabilized after the third subculture, whereas spore-bound Pr1 continues to decline following repeated subculturing. Decline in spore bound Pr1 was directly correlated with decline in virulence, however, such correlation with adhesion, hydrophobicity or surface charge could not be established. Because spore-bound Pr1 activities were directly correlated with M. anisopliae virulence; it could be used as a quality-control marker to monitor changes in virulence.

Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae)

Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain EAMa 01/121-Su against the German Cockroach, Blatella germanica (L.), was determined using four concentrations ranging from 4.2 × 10 6 to 4.2 × 10 9 spores per milliliter. The LD 50 value was 1.4 × 10 7 spores per milliliter (56,000 spores per cockroach) and LT 50 values were 14.8 days and 5.3 days for 4.2 × 10 8 and 4.2 × 10 9 spores per milliliter, respectively. An experiment was conducted to evaluate whether a fungal transmission could exist among infected and healthy cockroaches. Percentage mortality at a ratio of 1:10 of infected to unexposed cockroaches was 87.5% and LT 50 was 12.2 days, which indicated the potential of this strain to be horizontally transmitted and to rapidly spread the infection in the insect population. The effect of a sublethal dose (ca. LD 60) of M. anisopliae EAMa 01/121-Su strain, applied topically on German cockroaches, was studied by reciprocal crossing. Othecal production, oothecal hatchability, and nymphal production declined upon exposure to M. anisopliae EAMa 01/121-Su strain. The mean number of oothecae laid by female was progressively and significantly reduced by fungal treatment from second oviposition period onwards. Oothecal hatch of fungally challenged females was reduced by 46–49%, oothecal viability by 48–85%, and nymphal production by 22–35%. Only treated females showed an effect on oothecal production, oothecal hatch, and nymphal production, although oothecal hatch was also governed by treated males at a higher significance level. Our results on virulence and horizontal transmission of fungal conidia of M. anisopliae EAMa 01/121-Su strain and its sublethal reproductive effects on German cockroach females are discussed in terms of its potential to decrease the pest status of B. germanica in the short and long terms.

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 °C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 °C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.

Virulence of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) Strain ESC-1 to the German Cockroach (Dictyoptera: Blattellidae) and Its Compatibility with Insecticides

Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain ESC-1 against the German cockroach, Blattella germanica (L.), was determined using 5 concentrations ranging from 8 × 107 to 2 × 109 spores per milliliter. The calculated LD50 value was 4.18 by 108 spores per milliliter (4.18 × 105 spores per cockroach). In vitro study was conducted to determine the compatibility of M. anisopliae strain ESC-1 with chlorpyrifos, propetamphos, and cyfluthrin. Insecticides did not affect conidial germination but did adversely affect the growth and sporulation of M. anisopliae strain ESC-1. The growth of M. anisopliae colonies on media amended only with 50 and 500 ppm of chlorpyrifos and 500 ppm of propetamphos treatments at 3, 6, and 9 d was significantly inhibited compared with the control. Similarly, sporulation was significantly reduced in treated colonies exhibiting partial colony growth. The colonies cultured on SDAY media amended with 50 ppm of chlorpyrifos had significantly reduced sporulation compared with the control and no sporulation was observed in colonies cultured on media amended with 500 ppm of chlorpyrifos and propetamphos.