Development and testing of mycoinsecticides based on submerged spores and aerial conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) for control of locusts, grasshoppers and storage pests
Mycoinsecticides for pest control
Mykoinsektiziden für Schädlingsbekämpfung
by Adane Kassa
Date of Examination:2003-07-14
Date of issue:2003-09-17
Advisor:Prof. Dr. Stefan Vidal
Referee:PD Dr. Christian Borgemeister
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EnglishMicrobial control has recently been recognized as an important component of integrated pest management strategies for field crops and stored product commodities. The successful development of a mycoinsecticide ultimately depends on the availability of a virulent strain, an optimized and economic production system and a suitable formulation to optimise its application, efficacy and storage characteristics as well as the persistence after application. It is clear from analysis and synthesis of the present studies that considerable efforts have been made towards the selection of virulent isolates, the optimisation of the production process, the development of new formulations and testing their efficacy against the intended target insect pests, their persistence and storability under different environmental conditions. Accordingly, it was made possible to use BCAs for two distinct groups of agriculturally important insect pests, namely, field pests and storage insect pests. The field pest group was represented by locusts and grasshoppers (Locusta migratoria, Hieroglyphus daganensis and Cryptocatantops haemorrhoidalis), whereas the storage insect pests were represented by Sitophilus zeamais and Prostephanus truncatus. Due to the diverse nature of the test insects and their habitat, the methods followed to develop appropriate formulations are different for the two groups, for which the major findings are summarized as follows.The production and processing study indicates that submerged conidia of M. anisopliae var. acridum (IMI 330189) can be mass produced in a BH medium (3% biomalt and 1% yeast extract) under specific culturing conditions. Moreover, submerged conidia of M. anisopliae var. acridum (IMI 330189) can be effectively dried using the additives skimmed milk powder, molasses or glycerol either by freeze-drying or spray-drying technique. Storage at low temperatures (< 10°C) is possible for over 11 months without significant loss in viability. The initial viability of spores remains higher for spray-dried submerged spores. Furthermore, the viability and the efficacy of freeze-dried submerged conidia are significantly reduced when the product was ground to fine powder through a 120 µm mesh.When aerial conidia, submerged spores (mostly blastospores) and submerged conidia of M. anisopliae var. acridum (IMI 330189) were tested in different formulations in the laboratory and under field conditions, aerial conidia formulated in diesel oil displayed a higher efficacy against L. migratoria, H. daganensis and C. haemorrhoidalis at a lower application rate of 5 x 1012 conidia ha-1 than all submerged conidia and submerged spore formulations, which were applied at 1 x 1013 conidia ha-1. Freeze or spray dried submerged conidia of M. anisopliae var. acridum (IMI 330189) formulated in an oil flowable concentrate formulation are equally infectious to L. migratoria under laboratory conditions at a higher rate of 1 x 1013 spores ha-1.Submerged spores/conidia of M. anisopliae var. acridum (IMI 330189) were formulated as emulsions, as an oil flowable concentrate or as water-based formulations, out of which an oil flowable concentrate formulation revealed good biocontrol potential under laboratory and field conditions followed by emulsions. Under the conditions of the present research work and under the harsh environmental conditions that occur in the Sahel, water-based formulations of submerged spores are not suitable for grasshopper control using ULV applications. It was also observed that insects varied in their susceptibility to mycoinsecticide formulations, C. haemorrhoidalis being more susceptible to the mycopathogen than L. migratoria. Overall, the study provides evidence on the potential use of submerged conidia of M. anisopliae var. acridum under Sahelian environmental conditions. However, further improvements on the production process along with the quality of the formulation are indispensable.Strain selection according to thermal tolerance may be warranted when choosing an isolate for development as a microbial control agent. The results presented in this study confirm that there is a considerable variability in germination and growth at different temperatures among isolates of M. anisopliae, B. bassiana, B. brongniartii and Paecilomyces sp. The present study further demonstrated the susceptibility of S. zeamais and P. truncatus to non-specific entomopathogenic fungi indigenous to Ethiopia at varying degrees, P. truncatus being more susceptible to all isolates tested than S. zeamais. Furthermore, among the tested isolates, conidia and submerged spores/conidia of M. anisopliae (PPRC-EE) and B. bassiana (PPRC-HH,) appeared to be highly virulent to S. zeamais and P. truncatus. However, for the same isolates, the observed efficacy of submerged spores/conidia may or may not out way the aerial conidia and is entirely dependent on the type of liquid medium used for the production. The present study also identified one B. bassiana isolate (PPRC-HH), which produced an exceptionally higher yield of submerged conidia in TKI medium. Subsequently, M. anisopliae isolate (PPRC-EE) and B. bassiana isolate (PPRC-HH) were selected and various dustable powder (DP) formulations were developed and tested against stored product insect pests. The results revealed that all the tested formulations were active, (especially at higher concentration of 1 x 108 spores ml-1) when they were applied as a water suspension against S. zeamais. However, they show differences when they were incorporated on maize grains as a dry powder. Generally, DP formulations of aerial conidia were more effective against the two test insects than the corresponding submerged spore/conidia-based formulations. Moreover, aerial conidia-based formulations were more persistent, and could be stored 5 months at 4°C and 30°C than the corresponding submerged spore/conidia formulations. These storage characteristics could perfectly met under African conditions, where in most cases temperature in pesticide stores does not seem to exceed 30°C. Moreover, this study demonstrated that for dry powder formulations of submerged spores, a long term storage (up to 4 months) is possible at both 4°C and 30°C, however, a decline in viability and efficacy was observed at higher rates when the formulations were stored at 30°C for 5 months. This study also indicates the importance of inert carriers for improved efficacy, persistence and storability of DP formulations of B. bassiana and M. anisopliae spores.In general, the results of this study magnificently demonstrate that it is possible to achieve a successful level of control for S. zeamais and P. truncatus on stored and infested cereals using DP formulations of conidia of B. bassiana and M. anisopliae isolates. This would apparently be advantageous, especially in developing countries, where the risk of pesticide misuse is a major problem. Thus, the most effective formulations reported here could serve as a spring-board for further improvement of field application strategies. Furthermore, the fact that B. bassiana and M. anisopliae are easily amenable to large-scale production without a high input of technology essentially imply the feasibility of microbial control in the context of subsistence agriculture in developing countries such as Ethiopia. It can be inferred that developments being made now on the horizon would presumably make biological control of insect pests attractive from the practical point of view and also from an economic perspective in Ethiopia. This work could, therefore, be regarded as an important addition towards achieving this goal, as it would certainly shade-light to the sustainable development of more potent BCAs from the untapped beneficial microbial resources of the country.
Keywords: Biological control; entomopathogenic fungi; submerged spore; submerged conidia; aerial conidia; Beauveria bassiana; Metarhizium anisopliae; production; formulation; mycoinsecticides; locusts; grasshoppers; Locusta migratoria; Hieroglyphus daganensis; Cryptocatantops haemorrhoidalis; Sitophilus zeamais; Prostephanus truncatus; storage pest; spray-drying; Freeze-drying; dustable powder formulation
Im Rahmen des biologischen Pflanzenschutzes kommt dem Einsatz antagonistischer Mikroorganismen bei der Bekämpfung von Kultur- sowie von Lagerschädlingen eine besondere Bedeutung zu. Ein erfolgreicher Einsatz hängt aber maßgeblich von der Verfügbarkeit virulenter Isolate, geeigneter Produktions- und Formulierungsverfahren sowie von einer optimierten Applikation, Wirksamkeit, Lagerfähigkeit und Remanenz im Feld ab.In der vorliegenden Arbeit wurden Untersuchungen zur Entwicklung von Mykoinsektiziden gegen die Heuschreckenarten Locusta migratoria, Hieroglyphus daganensis und Cryptocatantops haemorrhoidalis sowie gegen die Lagerschädlinge Sitophilus zeamais und Prostephanus truncatus durchgeführt.Versuche zur Massenproduktion von Metarhizium anisopliae var. acridum (IMI 330189) ergaben, dass Submerskonidien in einem sog. BH medium (3 % Biomalz, 1 % Hefeextrakt) gebildet werden. Nach Zusatz von Milchpulver, Melasse oder Glycerin ist eine Gefrier- oder Sprühtrocknung möglich, wobei die Aktivität bei Lagertemperaturen unter 10°C über 11 Monate lang erhalten blieb.In Freilandversuchen in Afrika zeigte sich, dass in Dieselöl formulierte Luftkonidien nach Anwendung von 5 x 1012 Sporen/ha die beste Wirkung gegen die drei genannten Heuschreckenarten hatten. Im Labor waren dagegen gefrier- und sprühgetrocknete Submerskonidien, formuliert als Suspensionskonzentrat auf Ölbasis, gegen L. migratoria gleichwertig. Wässrige Formulierungen waren nicht wirksam.Bei der Auswahl von Isolaten gegen Lagerschädlinge konnten unterschiedliche Temperaturpräferenzen bei Isolaten von Metarhizium anisopliae, Beauveria bassiana, Beauveria brongniartii und Paecilomyces sp. nachgewiesen werden. Biotests mit diesen Isolaten ergaben, dass P. truncatus wesentlich empfindlicher reagierte als S. zeamais. Von zwei Isolaten (M. anisopliae PPRC-EE und B. bassiana PPRC-HH) wurden stäubbare Formulierungen sowohl von Luftkonidien als auch von Submerssporen entwickelt, die alle als wässrige Suspension sehr wirksam gegen S. zeamais waren. Nach Behandlung von Maiskörnern mit den Pulverformulierungen zeigten Luftkonidien verglichen mit Submerssporen eine bessere Wirkung und eine längere Lebensdauer nach 5 Monaten bei 4°C und 30°C.Die Ergebnisse haben gezeigt, dass eine biologische Bekämpfung von Heuschrecken und Lagerschädlingen mit Hilfe von entomopathogenen Pilzen und entsprechenden Formulierungen auch unter extremen Bedingungen möglich ist.
Schlagwörter: Biologische Schädlingsbekämpfung; Lagerschädlinge; Sitophilus zeamais; Prostephanus truncatus; Formulierung; Applikation; Massenproduktion; Submerskonidien; Submerssporen; Luftkonidien; gefriertrocken; sprühtrocken; Metarhizium anisopliae; Beauveria bassiana; Mykoinsektiziden; Formulierung; Pulverformulierungen; Heuschreckenarten; Locusta migratoria; Hieroglyphus daganensis; Cryptocatantops haemorrhoidalis