Pyroclastic material from the Puyehue-Cordon-Caulle Volcanic Complex, Chile, as carrier of Beauveria bassiana conidia: Potential utilization in mycoinsecticide formulations

Abstract

The last volcanic eruption of the Puyehue-Cordon-Caulle Volcanic Complex in the Andes cordillera of western South America, occurring on 4 June 2011, ejected pyroclastic materials that were accumulated in a wide region of the northern Patagonia (Argentina), affecting the environment and health of residents within the area. The aim of this work was to evaluate the practicability of using this waste material as a lowcost carrier for mycopesticide formulations. Beauveria bassiana is a recognized fungal agent for arthropod biologic control. Lengthy storage is critical for the development of mycoinsecticide formulations. Accordingly, the search for adequate materials to improve the shelf life of biocontrol products becomes desirable. First, several analytical techniques were employed to characterize the pyroclast physicochemically; then the viability of the fungal conidia was evaluated after an 18-month storage in the volcanic material. Finally, the pathogenicity of the conidia after that prolonged maintenance in the vehicle was assessed on the beetle Alphitobius diaperinus, an insect pest in poultry houses that causes major economic losses. The results from those bioassays proved auspicious for the eventual utilization of the pyroclast as a bioinsecticide carrier especially since the formulation had proven to be stable for at least 18 months under a wide range of environmental conditions. The constant moisture in a closed environment within a 5°C - 40°C temperature range insures a viable state during storage. The results indicate that what would otherwise be volcanic waste may be utilized as an efficient, abundant, inexpensive, and environmentally innocuous carrier of entomopathogenic fungi.

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S. Schalamuk, S. Pelizza, A. Scorsetti, M. González and I. Botto, "Pyroclastic material from the Puyehue-Cordon-Caulle Volcanic Complex, Chile, as carrier of Beauveria bassiana conidia: Potential utilization in mycoinsecticide formulations," Journal of Agricultural Chemistry and Environment, Vol. 3 No. 1, 2014, pp. 14-21. doi: 10.4236/jacen.2014.31003.

Figure 2.Grain-size-distribution curve. In the figure the percent size distribution is plotted versus the grain diameter in mm.

Figure 3.Reflection polarized light microscope images of the pyroclast pumice fragments showing (a) The shapes of the pu- mices; (b) Details of their porous surface.

Figure 4.SEM appearance of a typical pumicite grain.

Figure 5. Micro-Raman spectra registered in different point locations within the pyroclast.

The water loss proved variable, depending on the at- mospheric moisture. Differential-thermal-gravimetry stu- dies indicated two consecutive weight-loss signals in general: room temperature (RT)―80˚C and 80˚C - 200˚C, suggesting the presence of adsorbed water to different degrees. The pumiceous particles lost 7% of the adsorbed moisture at 50˚C, 14% at 100˚C and 18% at 200˚C. The hydric retention under adequate conditions can reach about 30%. A thermal stability between RT and 800˚C was indicated by X-ray diffraction, while the dehydration process (between the RT and ~100˚C) was reversible―a behavior similar to that observed in zeolite minerals.

Furthermore, the chemical analysis of a dried sample (Table 1) indicated the rhyolite-like composition cha- racteristic of pumiceous material, with an iron content (expressed as % Fe2O3) of 5.58. Notably, Ca, Mg, Na, K, Mn, Ba, Sr, and P were present as minor components.

3.2. Use of Volcanic Material as Vehicle of Beauveria bassiana Conidia

The use of dust carriers may improve the storage of the conidia as well as its distribution and application in the field [21]. Indeed, even under these conditions of nonrefrigeration for such a long period the viability of the B. bassiana (LPSC 1067) conidia was 97.4% ± 0.7% (where the average initial values were determined at 98.5 ± 0.8). An optimal shelf life of fungal spores under non- refrigerated storage conditions is of paramount practical- ity since mycopesticides may be exposed to high tem- peratures during transport, warehousing, or on-farm sto- rage [22-24]. Therefore, the very high viability deter- mined after this prolonged period of storage demonstra- tes that the pyroclast obtained after the recent PCCVC eruption is a promising geomaterial for use as a carrier for the biocontrol agent B. bassiana and possibly for other fungi.

The SEM micrograph of Figure 6(b) shows the dis- tribution and relative size of the conidia among the par- ticles of the inoculated volcanic ash after 18 months at room temperature. The conidia shown exhibit a certain morphologic deformation, probably an artifactual result of the preparation used for scanning electron microscopy since their viability was as high as 97% before the prep- aration of the samples for SEM.

Previous studies indicated that the spore longevity of entomopathogenic fungi was inversely proportional to temperature [25-27]. Blanford et al. [28] studied the in- teraction between temperature and humidity on the via- bility of nonformulated B. bassiana spores and found half-lifes of decay of 31, 49, and 71 days at 32˚C, 26˚C, and 22˚C, respectively, in environments with low relative humidities, but a slower decay in viability with half-lifes of 67 and 117 days at 26˚C and 22˚C, respectively, in environments with high relative humidities. Therefore, compared to those data with nonformulated spores, the present results showed that the volcanic material tested

Table 1. Major and minor elements (expressed as % oxides) by ICP-AES.

Figure 6. SEM micrograph of (a) The pyroclastic material without B. bassiana; (b) The same material impregnated with B. bassiana.

clearly favoured the prolongation of conidial viability. Since biocontrol products must remain stable for at least one year in order to be commercially acceptable [25]; the utilization of pyroclastic material could constitute a sta- ble, simple, and low-cost vehicle for B. bassiana formu- lations.

Because of the normal local seasonal variations occur- ring during the storage period, this formulation (i.e., geomaterial with bioinsecticide) was necessarily sub- jected to a substantial temperature variability (i.e., 5˚C - 40˚C). Nevertheless, this degree of variation did not af- fect the viability of B. bassiana conidia. A physico- chemical characterization of the matrix dust used under the conditions of experimental storage (i.e., the mass of the pyroclastic material and the volume of the receptacle) revealed an average weight loss of only 3% at up to 40˚C because the loss depended on the nature of the compo- nents of the pyroclastic mixture (0.5% for the crystalline phases and between 4% and 6% for the pumiceous par- ticles). Considerable disagreement exists in literature about the effect of moisture content on the viability of entomopathogenic fungal spores, a question that is even more complicated considering the influence of tempera- ture. Liquid water is cited as diminishing the viability of mycopesticide products by promoting spore germination [25]. Nevertheless, the decline of B. bassiana viability because of high temperatures can be significantly lower at high humidity environments [28]. The specific interac- tions with water that take place within the mass of this pyroclastic material may produce an environment with a suitable intermediate vapor pressure so as to protect the spores from dessication at high temperatures but still minimize the accumulation of liquid water that would necessarily cause a decline in viability. Therefore, the type of water in the material―i.e., that which is only adsorbed to the vesicles by a net physical process, thus leaving the material subject to a suitable vapor pressure (e.g., from water evaporation in a closed container de- pendent on the temperature, or by effect of the relative humidity of the environment)―may help prolong the longevity of spores.

Statistical analysis of the pathogenicity bioassays de- monstrated that the high percentages of mortality among the insects that were placed in the inoculated volcanic material after 18 months of storage (63.3% ± 3.5%) were significantly different (t = 2.31, p = 0.001) from the mortality of those exposed to the control substrate (8.31% + 0.7%; Figure 7). These results also indicated that con- tact of the insects with the ash alone produced only minimal mortality. In addition these same mortality deter- minations when performed on the conidia immediately after impregnation in the volcanic ash gave levels of 65.3% ± 2.5%, which values were statistically indistinguishable from the results represented in the figure (not shown). That level of mortality is high, compared to other studies evaluating the effects of B. bassiana on darkling beetles [29].

All the dead beetles in contact with the impregnated volcanic material exhibited an external growth of the fungus after a 24-h incubation in a humid chamber, thus demonstrating that death had been caused by mycosis (Figure 8). Darkling beetles represent one of the most pernicious pests in poultry production since they are known to transmit the viruses causing Marek’s disease, fowl pox, avian reovirus, infectious bursal disease, and Newcastle disease along with Aspergillus spp. and coc- cidia [12,13]. Because of the physical characteristics of the geomaterial tested, the ashes could be easily spread as a dust carrying B. bassiana for the biocontrol of A. diaperinus in poultry houses.

4. Conclusion

The results from bioassays on the killing efficiency of entomopathogenic fungi contained in volcanic materials from the PCCVC as a carrier proved auspicious for the

Figure 7. Percent mortality (means ± SD) of Alphitobius di- aperinus exposed to volcanic ash impregnated with Beauveria bassiana conidia and the volcanic-ash control. Values with different letter superscripts indicate significant differences ac- cording to the Student t-test.

Figure 8. Dead adults of Alphitobius diaperinus (Coleoptera: Tenebrionidae) infected by Beauveria bassiana conidia from impregnated volcanic ash after a 24-h incubation in a humid chamber.

eventual utilization of the pyroclast as a bioinsecticide vehicle. The biologic results in combination with the physicochemical properties of the carrier revealed that the formulation is stable for at least 18 months under a wide range of environmental conditions. We believe that the material tested can serve as an excellent substitute for other dust vehicles used to maintain the viability and pathogenicity of conidia of B. bassiana. In conclusion, this geomaterial would appear to be highly suitable for the preparation of formulations for pest control; thus utilizing what would otherwise be volcanic waste as an efficient, abundant, and inexpensive carrier of entomo- pathogenic fungi.

ACKNOWLEDGEMENTS

The work was done by financial support of ANPCyT BID 2011 PICT-2186 Argentina. Authors thank Lic. M. Theiller (CINDECA, University of La Plata) for SEM technical measurements. Dr. Donald F. Haggerty, a retired career investigator and native English speaker, edited the final version of the manuscript.

REFERENCES

[1] SERNAGEOMIN (2011) “Reportes especiales de act- ividad volcánica complejo volcánico puyehue―Cordón Caulle. www.sernageomin.cl

[2] Horwell, C.J. and Baxter, P. (2006) The respiratory health hazards of volcanic ash: a review for volcanic risk mitigation. Bulletin of Volcanology, 69, 1-24. http://dx.doi.org/10.1007/s00445-006-0052-y

[3] Knowles, D.A. (1998) Formulation of agrochemicals. In: Knowles, D.A., Ed., Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, The Netherland, 41-79. http://dx.doi.org/10.1007/978-94-011-4956-3_3

[4] Shah, P.A. and Pell, P.K. (2003) Entomopathogenic fungi as biological control agents. Applied Microbiology and Biotechology, 61, 413-423.

[5] Vega, F.E., Goettel, M.S., Blackwell, M., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzón, A., Ownley, B., Pell, J.K., Rangel, D. and Roy, H.E. (2009) Fungal entomopathogens: New insights on their ecology. Fungal Ecology, 2, 149-159. http://dx.doi.org/10.1016/j.funeco.2009.05.001

[6] Bhattacharyya, S. and Basu, M.K. (1982) Kaolin powder as a fungal carrier. Applied and Environmental Microbi- ology, 44, 751-753.

[7] Bidochka, M.J., Kamp, A.M. and De Cross, J.N.A. (2000) Insect pathogenic fungi: From genes to populations. In: Kronstad, J.W., Ed., Fungal Pathology, Springer, The Netherlands, 171-193.

[8] Jaronski, S.T. (2010) Ecological factors in the inundative use of fungal entomopathogens. BioControl, 55, 159-185. http://dx.doi.org/10.1007/s10526-009-9248-3

[9] Faria, M.R. and Wraight, S.P. (2007) Mycoinsecticides and mycoacaricides: A comprehensive list with world- wide coverage and international classification of formula- tion types. Biological Control, 43, 237-256. http://dx.doi.org/10.1016/j.biocontrol.2007.08.001

[10] Alves, L.F.A., Gassen, M.H., Pinto, F.G.S., Neves, P.M.O.J. and Alves, S.B. (2005) Natural occurrence of Beauveria bassiana (Bals.) Vuilleman (Moniliales: Moni- liaceae) on the lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), in a poultry house in Cascavel, PR. Neotropical Entomology, 34, 507-510. http://dx.doi.org/10.1590/S1519-566X2005000300021

[11] Daoust, R. and Roberts, D. (1983) Studies on the pro- longed storage of Metarhizium anisopliae conidia: Effect of temperature and relative humidity on conidial viability and virulence against mosquitoes. Journal of Invertebrate Pathology, 41, 143-150. http://dx.doi.org/10.1016/0022-2011(83)90213-6

[12] McAllister, J.C., Steelman, C.D., Newberry, L.A. and Skeeles, J.K. (1994) Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae). Journal of Medical Entomology, 31, 369-372.

[13] Goodwin, M.A. and Douglas Waltman, W. (1996) Trans- mission of Eimeria, viruses, and bacteria to chickens: Darkling beetles (Alphitobius diaperinus) as vectors of pathogens. The Journal of Applied Poultry Research, 5, 51-55.

[14] Pelizza, S.A., Eliades, L.A., Scorsetti, A.C., Cabello, M.N. and Lange, C.E. (2012) Entomopathogenic fungi from Argentina for the control of Schistocerca cancellata (Or- thoptera: Acrididae) nymphs: fungal pathogenicity and enzyme activity. Biocontrol Science and Technology, 22, 1119-1129. http://dx.doi.org/10.1080/09583157.2012.713910

[15] Pelizza, S.A., Eliades, L.A., Saparrat, M.C.N, Cabello, M.N., Scorsetti, A.C. and Lange, C.E. (2012) Screening of Argentine native fungal strain for biocontrol of the grasshoppers Tropidacris collaris: Relationship between fungal pathogenicity and chitinolytic enzyme activity. World Journal of Microbiology and Biotechnology, 28, 1359-1366. http://dx.doi.org/10.1007/s11274-011-0935-8

[16] Geden, C.J. and Steinkraus, D.C. (2003) Evaluation of three formulations of Beauveria bassiana for control of lesser mealworm and hide beetle in Georgia poultry hou- ses. Journal of Economic Entomology, 96, 1602-1607. http://dx.doi.org/10.1603/0022-0493-96.5.1602

[17] Lane, B.S., Humphreys, A.M., Thompson, K. and Trinci, A.P.J. (1988) ATP content of stored spores of Paecilo- myces farinosus and the use of ATP as criterion of spore viability. Transactions of the British Mycological Society, 90, 109-111. http://dx.doi.org/10.1016/S0007-1536(88)80186-4

[18] Haskin, L., Wang, A., Rockow, K., Jolliff, B., Korotev, R. and Viskupic, K. (1997) Raman spectroscopy for mineral identification and quantification for in situ planetary sur- face analysis: A point count method. Journal of Geo- physical Research, 102, 19293-19306. http://dx.doi.org/10.1029/97JE01694

[19] Das, S. and Hendry, M.J. (2011) Application of Raman spectroscopy to identify iron minerals commonly found in mine wastes. Chemical Geology, 290, 101-108. http://dx.doi.org/10.1016/j.chemgeo.2011.09.001

[20] Hanesch, M. (2009) Raman spectroscopy of iron oxides and (oxy)hydroxides at low laser power and possible ap- plications in environmental magnetic studies. Geophysi- cal Journal International, 177, 941-948. http://dx.doi.org/10.1111/j.1365-246X.2009.04122.x

[21] Ezzati-Tabrizi, R., Talaei-Hassanloui, R. and Pourian, H.R. (2009) Effect of formulating of Beauveria bassiana conidia on their viability and pathogenicity to the onion thrips, Thrips tabaci Lind. (Thysanoptera: Thripidae), 49, 97-104. http://dx.doi.org/10.2478/v10045-009-0013-5

[22] Hong, T.D., Ellis, R.H. and Moore, D. (1997) Develop- ment of a model to predict the effect of temperature and moisture on fungal spore longevity. Annals of Botany, 79, 121-128.

[23] Roberts, D.W. and Leger St., R.J. (2004) Metarhizium spp., cosmopolitan insect pathogenic fungi: Mycological aspect. Advances in Applied Microbiology, 54, 1-70. http://dx.doi.org/10.1016/S0065-2164(04)54001-7

[24] Faria, M., Hotchkiss, J.H. and Wraight, S.P. (2012) Ap- plication of modified atmosphere packaging (gas flushing and active packaging) for extending the shelf life of Beauveria bassiana conidia at high temperatures. Biolog- ical Control, 61, 78-88. http://dx.doi.org/10.1016/j.biocontrol.2011.12.008

[25] Jackson, M.A., Dunlap, C.A. and Jaronski, S.T. (2010) Ecological considerations in producing and formulating fungal entomopathogens for use in insect biocontrol. BioControl, 55, 129-145. http://dx.doi.org/10.1007/s10526-009-9240-y

[26] Hong, T.D., Gunn, J., Ellis, R.H., Jenkins, N.E. and Mo- ore, D. (2001) The effect of storage environment on the longevity of conidia of Beauveria bassiana. Mycological Research, 105, 597-602. http://dx.doi.org/10.1017/S0953756201004026

[27] Daoust, R.A., Ward, M.G. and Roberts, D.W. (1983) Ef- fect of formulation on the viability of Metarhizium ani- sopliae conidia. Journal of Invertebrate Pathology, 41, 151-160. http://dx.doi.org/10.1016/0022-2011(83)90214-8

[28] Blanford, S., Jenkins, N.E., Christian, R., Chan, B.H.K., Luisa, N., Michael, O., Koekemoer, L., Coetzee, M., Read, A.F. and Thomas, M.B. (2012) Storage and persis- tence of a candidate fungal biopesticide for use against adult malaria vectors. Malaria Journal, 11, 354. http://dx.doi.org/10.1186/1475-2875-11-354 http://www.malariajournal.com/content/11/1/354

[29] Gindin, G., Glazer, I., Mishoutchenko, A. and Samish, M. (2009) Entomopathogenic fungi as a potential control agent against the lesser mealworm, Alphitobius diaperi- nus in broiler houses. BioControl, 54, 549-558. http://dx.doi.org/10.1007/s10526-008-9205-6

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] SERNAGEOMIN (2011) “Reportes especiales de actividad volcánica complejo volcánico puyehue—Cordón Caulle. www.sernageomin.cl
[2] Horwell, C.J. and Baxter, P. (2006) The respiratory health hazards of volcanic ash: a review for volcanic risk mitigation. Bulletin of Volcanology, 69, 1-24. http://dx.doi.org/10.1007/s00445-006-0052-y
[3] Knowles, D.A. (1998) Formulation of agrochemicals. In: Knowles, D.A., Ed., Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, The Netherland, 41-79.
http://dx.doi.org/10.1007/978-94-011-4956-3_3
[4] Shah, P.A. and Pell, P.K. (2003) Entomopathogenic fungi as biological control agents. Applied Microbiology and Biotechology, 61, 413-423.
[5] Vega, F.E., Goettel, M.S., Blackwell, M., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzón, A., Ownley, B., Pell, J.K., Rangel, D. and Roy, H.E. (2009) Fungal entomopathogens: New insights on their ecology. Fungal Ecology, 2, 149-159. http://dx.doi.org/10.1016/j.funeco.2009.05.001
[6] Bhattacharyya, S. and Basu, M.K. (1982) Kaolin powder as a fungal carrier. Applied and Environmental Microbiology, 44, 751-753.
[7] Bidochka, M.J., Kamp, A.M. and De Cross, J.N.A. (2000) Insect pathogenic fungi: From genes to populations. In: Kronstad, J.W., Ed., Fungal Pathology, Springer, The Netherlands, 171-193.
[8] Jaronski, S.T. (2010) Ecological factors in the inundative use of fungal entomopathogens. BioControl, 55, 159-185. http://dx.doi.org/10.1007/s10526-009-9248-3
[9] Faria, M.R. and Wraight, S.P. (2007) Mycoinsecticides and mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biological Control, 43, 237-256. http://dx.doi.org/10.1016/j.biocontrol.2007.08.001?
[10] Alves, L.F.A., Gassen, M.H., Pinto, F.G.S., Neves, P.M.O.J. and Alves, S.B. (2005) Natural occurrence of Beauveria bassiana (Bals.) Vuilleman (Moniliales: Moniliaceae) on the lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), in a poultry house in Cascavel, PR. Neotropical Entomology, 34, 507-510. http://dx.doi.org/10.1590/S1519-566X2005000300021
[11] Daoust, R. and Roberts, D. (1983) Studies on the prolonged storage of Metarhizium anisopliae conidia: Effect of temperature and relative humidity on conidial viability and virulence against mosquitoes. Journal of Invertebrate Pathology, 41, 143-150. http://dx.doi.org/10.1016/0022-2011(83)90213-6
[12] McAllister, J.C., Steelman, C.D., Newberry, L.A. and Skeeles, J.K. (1994) Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae). Journal of Medical Entomology, 31, 369-372.
[13] Goodwin, M.A. and Douglas Waltman, W. (1996) Transmission of Eimeria, viruses, and bacteria to chickens: Darkling beetles (Alphitobius diaperinus) as vectors of pathogens. The Journal of Applied Poultry Research, 5, 51-55.
[14] Pelizza, S.A., Eliades, L.A., Scorsetti, A.C., Cabello, M.N. and Lange, C.E. (2012) Entomopathogenic fungi from Argentina for the control of Schistocerca cancellata (Orthoptera: Acrididae) nymphs: fungal pathogenicity and enzyme activity. Biocontrol Science and Technology, 22, 1119-1129.
http://dx.doi.org/10.1080/09583157.2012.713910
[15] Pelizza, S.A., Eliades, L.A., Saparrat, M.C.N, Cabello, M.N., Scorsetti, A.C. and Lange, C.E. (2012) Screening of Argentine native fungal strain for biocontrol of the grasshoppers Tropidacris collaris: Relationship between fungal pathogenicity and chitinolytic enzyme activity. World Journal of Microbiology and Biotechnology, 28, 1359-1366. http://dx.doi.org/10.1007/s11274-011-0935-8
[16] Geden, C.J. and Steinkraus, D.C. (2003) Evaluation of three formulations of Beauveria bassiana for control of lesser mealworm and hide beetle in Georgia poultry houses. Journal of Economic Entomology, 96, 1602-1607. http://dx.doi.org/10.1603/0022-0493-96.5.1602
[17] Lane, B.S., Humphreys, A.M., Thompson, K. and Trinci, A.P.J. (1988) ATP content of stored spores of Paecilomyces farinosus and the use of ATP as criterion of spore viability. Transactions of the British Mycological Society, 90, 109-111. http://dx.doi.org/10.1016/S0007-1536(88)80186-4
[18] Haskin, L., Wang, A., Rockow, K., Jolliff, B., Korotev, R. and Viskupic, K. (1997) Raman spectroscopy for mineral identification and quantification for in situ planetary surface analysis: A point count method. Journal of Geophysical Research, 102, 19293-19306. http://dx.doi.org/10.1029/97JE01694
[19] Das, S. and Hendry, M.J. (2011) Application of Raman spectroscopy to identify iron minerals commonly found in mine wastes. Chemical Geology, 290, 101-108. http://dx.doi.org/10.1016/j.chemgeo.2011.09.001
[20] Hanesch, M. (2009) Raman spectroscopy of iron oxides and (oxy)hydroxides at low laser power and possible applications in environmental magnetic studies. Geophysical Journal International, 177, 941-948. http://dx.doi.org/10.1111/j.1365-246X.2009.04122.x
[21] Ezzati-Tabrizi, R., Talaei-Hassanloui, R. and Pourian, H.R. (2009) Effect of formulating of Beauveria bassiana conidia on their viability and pathogenicity to the onion thrips, Thrips tabaci Lind. (Thysanoptera: Thripidae), 49, 97-104. http://dx.doi.org/10.2478/v10045-009-0013-5
[22] Hong, T.D., Ellis, R.H. and Moore, D. (1997) Development of a model to predict the effect of temperature and moisture on fungal spore longevity. Annals of Botany, 79, 121-128.
[23] Roberts, D.W. and Leger St., R.J. (2004) Metarhizium spp., cosmopolitan insect pathogenic fungi: Mycological aspect. Advances in Applied Microbiology, 54, 1-70. http://dx.doi.org/10.1016/S0065-2164(04)54001-7
[24] Faria, M., Hotchkiss, J.H. and Wraight, S.P. (2012) Application of modified atmosphere packaging (gas flushing and active pack-aging) for extending the shelf life of Beauveria bassiana conidia at high temperatures. Biological Control, 61, 78-88. http://dx.doi.org/10.1016/j.biocontrol.2011.12.008
[25] Jackson, M.A., Dunlap, C.A. and Jaronski, S.T. (2010) Ecological considerations in producing and formulating fungal entomopathogens for use in insect biocontrol. BioControl, 55, 129-145. http://dx.doi.org/10.1007/s10526-009-9240-y
[26] Hong, T.D., Gunn, J., Ellis, R.H., Jenkins, N.E. and Moore, D. (2001) The effect of storage environment on the longevity of conidia of Beauveria bassiana. Mycological Research, 105, 597-602.
http://dx.doi.org/10.1017/S0953756201004026
[27] Daoust, R.A., Ward, M.G. and Roberts, D.W. (1983) Effect of formulation on the viability of Metarhizium anisopliae conidia. Journal of Invertebrate Pathology, 41, 151-160. http://dx.doi.org/10.1016/0022-2011(83)90214-8
[28] Blanford, S., Jenkins, N.E., Christian, R., Chan, B.H.K., Luisa, N., Michael, O., Koekemoer, L., Coetzee, M., Read, A.F. and Thomas, M.B. (2012) Storage and persistence of a candidate fungal biopesticide for use against adult malaria vectors. Malaria Journal, 11, 354. http://dx.doi.org/10.1186/1475-2875-11-354 http://www.malariajournal.com/content/11/1/354
[29] Gindin, G., Glazer, I., Mishoutchenko, A. and Samish, M. (2009) Entomopathogenic fungi as a potential control agent against the lesser mealworm, Alphitobius diaperinus in broiler houses. BioControl, 54, 549-558. http://dx.doi.org/10.1007/s10526-008-9205-6

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