Conditions to optimize mass production of Metarhizium anisopliae Metschn. Metarhizium anisopliae Metschn. Sorokin Hypocreales: Clavicipitaceae is an entomopathogenic fungus with great potential as a biological pesticide to biologically control pests. However, the relatively high cost of the substrate needed for its mass production system increases product price and discourages its use.
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It is most frequently applied to berry fruit crops, where fungicides are also used for disease control. Fungicides: azoxystrobin, benomyl, captan, chlorothalonil, fenhexamid, fludioxonil, iprodione, and metalaxyl in concentrations of 0. Those strains were selected because of their present use against different insect pest in bramble fruits. Results indicate that the benomyl and fenhexamid fungicides were compatible with the five isolates whereas, azoxystrobin and fludioxonil were incompatible.
Key words: biological control, integrated management, fungal growth, conidia germination. Los resultados indicaron que los fungicidas benomyl y fenhexamid fueron compatibles con las cinco cepas, mientras que azoxystrobin y fludioxonil fueron incompatibles con todas las cepas evaluadas. Among the most studied insect pathogenic fungi species are Hyphomycetes Beauveria bassiana , Metarhizium anisopliae Metschnikoff Sorokin, and Verticillium lecanii Khetan, EF efficiency can be affected by numerous coactions of biotic and abiotic factors: desiccation, UV light, host behavior, temperature, entomopathogen vigor, host condition, and agrochemical effects Flexer and Belnavis, ; Inglis et al.
Plant protection products acting on EF can vary as a function of the species and EF structure type, chemical nature of the product, and doses used.
Effects decrease vegetative growth and germination, as well as genetic mutations, virulence, including EF death Alves et al.
Fungicides are among the products frequently used in agriculture that would theoretically act directly on EF. However, there is background information about the different degrees of EF fungicide tolerance.
Rebollar et al. Using EF to control important agricultural pests is relatively recent in Chile and its application has emphasized berry pest control in both organic and conventional management Devotto and Gerding, Therefore, there are cases where applying EF is prior to or follows the application of frequently used fungicides to control disease and which are registered for berries.
It is not known what effect these products can have on EF which are actually being used for biological control of berry pests such as multiple strains of Metarhizium anisopliae. Consequently, the objective of this study was to determine the effect of allowed fungicides in berries on conidia germination and vegetative growth of the M. Strain selection and multiplication Five frequent strains of M. Conidia were then collected with a bacteriological loop and superficially spread on 80 mm diameter Petri dishes with PDA medium.
At the end of this incubation period, fungal material obtained was employed to evaluate the relative growth of the colonies. The plating procedure was carried out under sterile conditions in a laminar flow chamber. Table 1. Origin latitude and longitude and pest controlled by Metarhizium anisopliae var. Effect of fungicides on the relative growth of colonies The preparation of media was carried out in flasks with a concentration of 39 g L -1 PDA; agar was dissolved in a microwave for 10 min and then sterilized for 20 min.
PDA supplemented with distinct fungicide concentrations was poured onto Petri dishes 80 mm diameter and once the medium solidified, a 5 mm diameter cylinder was extracted from the center of each dish with a lab scoop. Disks with a 5 mm diameter were extracted from the edge of the growth of incubated dishes, during the strain multiplication stage, and when the colony advanced up to half the dish radius.
These disks replaced a cylinder of the same size, like the one taken from the dishes containing PDA supplemented with fungicides. Dishes were incubated in the same conditions as in the previous stage. Colony diameter was measured every 48 h on four equidistant radii previously marked on the bottom of each dish.
Relative growth of the colonies RG was calculated from the data for each strain, fungicide, and concentration. Subsequently, the growth rate for each active ingredient, concentration, and strain was calculated for these colonies. A completely randomized experimental design with three replicates for each fungicide strain and concentration was applied Fry, Effects of fungicides on conidia germination and germination tube length Growth rates for each isolate, fungicide, and concentration were calculated with colony diameter data.
A linear regression with the OrigenPro software was carried out on the data, adjusting two types of curves sigmoid and exponential depending on the different strain responses to distinct active ingredients Fry, Similarly, a response curve and associated equation were obtained for each isolate and fungicide.
A 1 mL conidia suspension of each strain was then inoculated, homogeneously distributed on Petri dishes with PDA supplemented with the IC50 corresponding to each strain and fungicide, whereas the control was a Petri dish with only PDA.
Dishes were incubated in the same conditions as in the previous assays. A germination count was then carried out considering only those conidia that exhibited germination tube lengths equal to or greater than conidia length.
Results were expressed as a percentage of germination. This assay had a completely random design with three replicates, and the experimental unit consisted of conidia. To normalize germination percentage data before ANOVA, an angular transformation was carried out according to the methodology used by Fry The shape of the curves for all isolates addressing this active ingredient were similar, growth decreased as the concentration of active ingredient increased Figure 1 A.
Azoxystrobin belongs to the strobilurin chemical group and accomplishes its fungal action by inhibiting the mitochondrial respiration of fungi, blocking electron transport between cytochrome b and cytochrome c 1 where ubiquinol coenzyme Q is oxidized, directly interfering with the oxidative phosphorylation process since there would be no more available energy in the mitochondria to synthesize ATP adenosine triphosphate , and impeding spore germination and pathogen development Ypema and Gold, ; Agrios, ; Kanetis et al.
There would therefore be arguments to point out that a lack of energy can affect vegetative development in the studied isolates. Furthermore, there are reports indicating that azoxystrobin is incompatible with EF B.
Figure 1. Colony relative growth RG of Metarhizium anisopliae var. Figure 2. It must be mentioned that comparing AUGIC allows contrasting isolate response with the evaluated fungicides; however, these are only another indicator since the shape of each curve is different and with distinct extreme values for each one of them.
Therefore, in addition to the value of AUGIC, it is also important to know the shape of the curve from which this value was obtained. The Qu-M82 isolate was incompatible beginning with 1 mg L -1 concentration and all the rest were incompatible starting from the 10 mg L -1 concentration.
Hence, all isolates are affected at concentrations equal to or greater than 10 mg L -1 and almost entirely inhibit growth Figure 1 B. Benomyl is in the benzimidazol group nitro organic heterocyclic characterized for inhibiting tubulin and impeding normal mitosis through the modification of the fungus cellular mechanism by competition with the free puric bases adenine and guanine or with formed nucleotides Agrios, However, it is recognized that fungi exposed to this fungicide can suffer mutations that prevent the inhibitory effect of tubulin, mutations that can be found in nature, thus explaining the distinct growth values in M.
Similar results have been reported by Tedders and Alves et al. Tamai et al. On the other hand, Qu-M was incompatible beginning with the 0. Captan is part of the dicarboximide group which impedes fungi respiration by interfering with Krebs cycle along with the inhibitory activity of sulfhydryl enzymes Agrios, These arguments could explain isolate incompatibility for this active ingredient.
Chandler and Davidson reported the inhibitory action of chlorothalonil on M. This active ingredient is part of the chemical group of substituted aromatics benzene derivative and isoftalonitril acting on enzymes and hormones by inhibiting sulfhydryl radicals and other peptides, such as glutathione, during the germination process, disrupting glycolysis, and energy production Agrios, However, Alves et al.
This fungicide has also been reported to be compatible with other fungi such as B. Fenhexamid was incompatible with all the isolates in the mg L -1 concentration Figure 1 E. It is possible that this action mechanism has affected the evaluated strains; however, there are no known reports of the effect of fenhexamid on EF. All M. To date, no reports have been found on the action of fludioxonil on EF. This fungicide is characterized by its increase in glycerol synthesis intercellular osmotic pressure regulating substance , provoking hypertrophy and blocking fungus cellular growth, a mechanism which could also have taken place in the M.
The rest of the isolates were incompatible only beginning with the 10 mg L -1 concentration of iprodione Figure 1 G. These results coincide with those reported by Chandler and Davidson who point out that iprodione is incompatible with M. The rest of the isolates were incompatible with all the evaluated concentrations Figures 1 H and 2 H.
Although metalaxyl is not recommended to control hyphomycetes, in this case, an incompatibility effect of the product with colony growth was observed. Loureiro et al. The mode of action of metalaxyl is to impede protein formation by interfering with the biosynthesis of nucleic acids and inhibit polymerase enzyme action in ribonucleic acid synthesis Agrios, It is probable that this action mechanism can occur in fungi cultivated in vitro , but not necessarily for in situ conditions.
Effect of fungicides on conidia germination and germination tube length It was possible to calculate the value of IC50 only for all the isolates incubated with benomyl, and only for Qu-Mb in the case of fludioxinil. The latter reached an IC50 of 0. Table 2. Benomyl inhibitory concentration 50 IC50 of Metarhizium anisopliae var. Isolate germination percentages decreased in all cases from Figure 3. Conidia germination A and germinative tube lengths B of Metarhizium anisopliae var.
The response of isolates treated with benomyl was different when germination tube length was evaluated. The fact that benomyl IC50 has affected normal germination development can be due to the mode of action of this active ingredient which was previously mentioned Agrios, This inhibition probably generated a decrease in conidia germination and a decrease in germination tube length in those able to germinate, which in turn explains a decline in vegetative growth.
The action mechanism of fludioxonil Agrios, ; Kanetis et al. Figure 4. The natural variability between isolates of the same species of EF is extensively described in the literature for biological parameters such as pathogenicity on an organism. Becerra et al. Three of the five evaluated isolates in this bioassay were characterized by the aforementioned authors who concluded that the Qu-Mb, Qu-M, and Qu-M isolates belong to the same genetic group; however, Qu-M and Qu-M would be more genetically similar than Qu-Mb.
This variability could also explain sensitivity to synthetic chemical products Alves et al. Furthermore, Inglis et al. This is evidenced by observing the different responses among isolates as compared to the fungicides evaluated in this bioassay. Azoxystrobin and fludioxonil were incompatible and therefore should be avoided in integrated pest management when some of these M. The captan, chlorothalonil, iprodione, and metalaxyl fungicides showed different responses, so their action cannot be generalized for this entomopathogenic fungus.
Fludioxonil and benomyl decreased conidia germination of evaluated M. Plant pathology. Moino, e J. Alves ed.
Insect pathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase MLS and isocitrate lyase ICL , with particular attention to the glyoxylate cycle when M. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds acetate and ethanol as well as in insect haemolymph. We fused the promoter of the M. Furthermore, Ma-icl was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation.