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The effect of fatty, organic or phenolic acids on the germination of conidia of Metarhizium flavoviride
Mycol. Res. 101 (6) : 662–666 (1997)
662
Printed in Great Britain
The effect of fatty, organic or phenolic acids on the germination of conidia of Metarhizium flavoviride
S. E. B A R N E S A N D D. M O O R E International Institute of Biological Control, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7TA, U.K.
Conidia of Metarhizium flavoviride were suspended in oil formulations and received fatty and other acids before germination. This was done by the acids being added either to the conidial suspensions before inoculation onto agar plates or directly into the agar before the conidia were applied. Certain acids of chain lengths C10 and below, including lactic, salicylic, caprylic and capric acids produced significant inhibition of germination when added to the oil formulations. When incorporated into the agar, all acids of C10 and below were inhibitory, with the exception of sebacic and succinic acids. Sebacic acid (a C10 saturated dicarboyxlic acid) and all the longer chain fatty acids sampled produced no inhibition of germination, either when added to the formulation or the agar. Tests were carried out on a sample of conidia which showed no germination ; a few acids stimulated some germination. These included sebacic acid and longer chain fatty acids such as stearic and (inconsistently) lauric acids. Conidia from a long term storage experiment showed increased germination on the addition of stearic and}or lauric acids. The inhibitory effect of capric acid on germination was overcome by a 4–15 times higher concentration of stearic acid.
The use of Metarhizium flavoviride for the biological control of locusts and grasshoppers is being researched in a collaborative project involving the International Institute of Biological Control (IIBC), the International Institute of Tropical Agriculture (IITA), Cotonou, in the Republic of Benin and the De! partement de Formation en Protection des Ve! ge! taux (DFPV), Niamey, Niger. The conidia of M. flavoviride are lipophilic and can be formulated in oil for application with ultra-low volume equipment. Formulation in oil increases efficacy and reduces dependence on humid conditions (Prior et al., 1988 ; Bateman et al., 1993) ; field trials have been successful in killing locusts and grasshoppers, but the speed of kill is slower than desired and research is being undertaken to reduce time to death. The formulation and storage of M. flavoviride conidia in oil is logistically easy. However, storage of conidia in different oils results in variable germination (Stathers et al., 1993 ; Moore et al., 1995) ; one factor causing this may be the presence of free fatty acids in the oils, some of which have been reported to inhibit, and others promote, germination of fungal conidia (Kerwin, 1987 ; Bidochka & Khachatourians, 1992). Most vegetable oils contain amongst others, palmitic, oleic and linoleic acids (Ro$ bbelen et al., 1989). Components of the cuticle of an insect may have both inhibitory and nutritional effects on the germination of conidia including, Beauveria bassiana and M. anisopliae ; these effects could be due, in part, to the fatty acids present on the cuticle (Lecuona et al., 1991; Butt et al., 1995). For Schistocerca gregaria (Forska/ l) these fatty acids occur at different levels, (C16 " C14 " C10 " C12 " C18) with 1±5 % of the cuticle’s
composition being free fatty acids for females and 4±7 % for males (Oraha & Lockey, 1990). Some fatty acids may stimulate the initiation of germination and encourage hyphal growth, whilst others may inhibit both these processes. Oleic and linoleic acids increase, whilst stearic and linolenic inhibit, mycelial growth of Beauveria bassiana (Bidochka and Khachatourians, 1992). Conidia of Nomuraea rileyi have been shown to rely completely on an exogenous supply of fatty acids and other substrates for germination (Boucias et al., 1984). Short chain fatty acids block the uptake of phosphate and thiamine in certain fungi, preventing the conidia from germinating (Kerwin, 1987). Fungi have fatty acid carrier systems which effect selective uptake through fungal membranes according to carbon chain length (Kerwin, 1987). This may influence which fatty acids inhibit and which promote germination. The major fungal fatty acids are unbranched C12 to C20 chains, mostly even numbered, the main examples in fungi being palmitoleic, oleic, linoleic and linolenic (Kerwin, 1987). In the present work we investigated effects of various fatty acids on the germination of conidia of M. flavoviride to determine if any means of hastening or increasing germination, and hence increasing the speed of kill, could be found. MATERIALS AND METHODS Fungal material An isolate of M. flavoviride (Metschn.) Sorokı- n(IMI 331089) had been obtained from Ornithacris cavroisi (Finot) (Orthoptera : Acrididae) at Niamey, Niger in 1988. Two replicate samples
S. E. Barnes and D. Moore of conidia (I & II), which had been dried to around 5 % moisture content and mixed with silica gel, were obtained after 10 months storage at 10 °C. One gram of each of these replicates was suspended in 200 ml of a 50}50 mix of two mineral oils used in pesticide applications, Shellsol, a light oil, and Ondina, a more viscous oil. The conidial suspensions (of approx. 2±5¬10) conidia ml−") were sonicated (Lucas Dawe Ultrasonicater Sonicleaner) for 3 min before being stored at 10° C. Other suspensions, of the same isolate, were taken from a long term experiment (Moore et al., 1995), the spores having been stored for 4 years at 8° in several oil mixtures. Fatty acids Twenty-two samples of various acids (Croda Chemicals Ltd, Goole, John L. Seaton and Co Ltd, Hull and Sigma Ltd, Poole) were tested for their effects on the germination of M. flavoviride. These included organic acids such as citric and salicylic, fatty acids, including dicarboxylic fatty acids such as succinic and a phenolic acid, eugenol. Only linoleic acid and eugenol were supplied by Sigma. Four fatty acids (C12, lauric ; C14, myristic : C16, palmitic and C18, stearic), came in two forms either named, or labelled by their carbon number ; these samples varied slightly in their commercial processing but were all of 95 % purity or better with impurities being of closely related acids. To distinguish the two forms of fatty acids specified above, samples labelled only by C numbers when supplied are referred by their C number followed by name eg C12 (lauric) in the text. Those named are referred to as, e.g., lauric (C12) in the text. Solutions of most of the fatty acids were initially made up to 2 % concentrations and added to an equal volume of oil formulation of conidia, to reduce the fatty acid concentration to 1 %. Some of the fatty acids were not soluble in the oil solutions at the higher concentrations ; in these cases a final concentration of 0±75 % fatty acid in the oil formulation was obtained (Table 1). In certain cases the oil was warmed to dissolve the acid. The control treatment was the conidial suspension with no added acids.
663 Table 1. Germination (mean percentage³CI) of Metarhizium flavoviride conidia in oil formulations. Fatty acids and other materials were either added to the oil formulation or incorporated into the agar (Sample I) Fatty acid added to formulation
Control Lactic Succinic Tartaric Adipic Citric Salicylic Caprylic Capric Eugenol Sebacic C12 (Lauric) Lauric C14 (Myristic) Myristic C16 (Palmitic) Palmitic C18 (Stearic) Stearic Linoleic Oleic Behenic Erucic
Fatty acid added to agar
% added
Mean
³95 % CI
Mean
³95 % CI
— 1 0±75 0±75 0±75 0±75 0±75 0±75 0±75 1 0±75 1 1 1 0±75 1 0±75 1 0±75 1 1 0±04 1
84 5 84 87 84 61 16 0 0 3 85 87 81 86 83 83 86 86 86 89 89 85 83
8±8 13±4 7±3 5±1 8±1 38±9 9±0 — — 5±5 8±1 7±7 19±9 3±7 18±9 12±9 6±0 0±6 3±1 1±1 4±8 4±2 10±6
88 0±4 86 35 27 66 0 0 0 0 65 73 86 89 86 91 88 89 79 NA 89 89 81
4±2 1±0 11±2 17±0 24±8 20±0 — — — — 38±0 27±8 4±1 3±9 7±5 4±3 9±2 4±5 13±4 NA 5±9 4±8 35±0
NA, not available. It was not possible to obtain a count from these plates as the agar was clouded ; growth was seen on the plates after several days.
for each treatment of suspension I at 24 h and from three for sample II at 24 and 44 h. Experiment 2 : Germination counts on plates containing fatty acids Acids were added to molten SDA, and mixed before been poured onto plates which were inoculated with conidial suspension I. Germination counts were carried out on five plates per treatment assessed after 24 h at 25°.
Viability test The percentage germination was determined by spreading 0±1 ml of each oil suspension on SDA plates (Sabouraud Dextrose Agar) (Oxoid, Basingstoke) with a small spatula. The plates were incubated at 25°, usually for 24 h but for 44 h where conidia were slower to germinate. The plates were scanned at ¬300 magnification, and at least 300 conidia were assessed on each plate. Single conidia were assessed to avoid difficulties in determining the source of germ tubes in clumps of conidia. Germination was considered to have occurred when the germ tubes projected from the conidia to a length approx half that of the length of the conidia. Experiment 1 : Percentage germination after inoculation with fatty acids Acids were added to conidial suspensions I and II as outlined above. Germination counts were carried out from five plates
Experiment 3 : Fatty acids on previously stored samples Germination counts were carried out on 21 samples of M. flavoviride in a number of oils from long term storage experiments (Moore et al., 1995). Stearic acid (C18) at 0±5 % and C12 (lauric) at 1 % concentration were added to equal volumes of conidial formulating resulting in 0±25 and 0±5 % concentrations respectively. Five plates were assessed for germination from each treatment after 24 h at 25°. Experiment 4 : Tests using both an inhibitory and a stimulatory fatty acid Suspension I was combined with both capric acid, which had been shown to inhibit germination, and stearic acid (C18), either separately or together. The treatments started as a 1 % concentration of capric and}or stearic acid in an oil formulation of conidia. The combined treatments of formulation : stearic :
Effects of fatty acids on germination of conidia of M. flavoviride capric began at the ratio of 100 : 1 : 1. Capric acid concentrations were decreased in subsequent treatments, down to a 100 : 1 : 0±063 ratio. Five replications were carried out for each treatment.
Table 3. Germination of Metarhizium flavoviride conidia stored in a range of oils for 4 years with or without added C12 (lauric) and}or stearic (C18) fatty acids
Formulation
Formulation C12
Formulation C12 stearic
Oil used
Mean (%)
³..
Mean (%)
³..
Mean (%)
³..
GSSAO SSSAO ESS GDK GSS SDKAO GDKAO EDK SDK SSS GSSAO SSSAO ESS GDK GSS SDKAO GDKAO EDK SDK SSS
52 71 81 83 80 16 74 87 74 62 65 78 85 89 90 52 81 85 75 75
3±0 3±6 2±3 2±4 2±6 1±1 1±5 2±5 2±6 4±4 2±2 2±9 1±7 1±1 0±7 6±1 1±6 3±5 2±8 4±5
56 74 85 88 85 18 83 90 80 67 75 77 86 92 91 59 89 89 79 80
3±8 2±9 1±4 1±5 3±5 2±4 2±7 1±5 1±6 2±7 1±4 4±9 1±1 0±4 1±4 2±9 0±8 1±7 3±4 2±6
66 80 88 92 91 22 83 93 86 76 77 84 90 92 95 67 91 92 85 87
3±0 4±0 2±5 1±8 1±6 1±8 1±4 1±2 3±1 4±3 2±9 1±9 0±5 0±8 0±7 4±5 0±7 0±5 1±6 1±9
RESULTS The results of treatments to sample I in experiment 1 (Table 1), showed that the addition of most fatty acids did not reduce germination compared with the control level of 84±1 %. However, caprylic and capric acids, when added to the oil formulation, completely inhibited germination, while additionally eugenol and salicylic were completely inhibitory when included in the agar treatment. Other treatments also caused marked inhibition of germination when added to the oil formulation or agar plates. Citric, sebacic and C12 (lauric) acids may have been mildly inhibitory as replicates results were variable, indicated by high confidence intervals. Little or no effect on percentage germination was demonstrated by succinic, sebacic (added to oil) and C12 or longer chain acids. The high germination rate of the control did not lend itself to showing substances which promote germination. There was no significant difference in the effects on germination caused by the fatty acids in the two different treatment methods, oil and agar, except for adipic (F , ¯ 36±59 ; P ! 0±001), salicylic ") (F , ¯ 6±05 ; P ! 0±05) and tartaric acids (F , ¯ 67±28 ; P ! ") ") 0±001). In each case germination on agar plus acid was significantly lower than that of conidia in oil plus acid. In contrast, sample II showed no control germination, but the addition of some acids stimulated germination (Table 2), although usually at low levels. Only eight treatments resulted Table 2. Germination of Metarhizium flavoviride conidia in oil formulation after adding fatty acids and other materials and incubating for 24 or 44 h (Sample II) 24 h
Control Lactic Succinic Tartaric Adipic Citric Salicylic Caprylic Capric Eugenol Sebacic C12 (Lauric) Lauric C14 (Myristic) Myristic C16 (Palmitic) Palmitic C18 (Stearic) Stearic Linoleic Oleic Behenic Erucic
44 h
Mean (%)
³..
Mean (%)
³..
0 0 0±3 0 1 0±1 1 0 0 0 13 10 0 1 5 0±3 0 1 2 3 0 1 0
— — 0±3 0±0 1±1 0±1 0±5 — — — 3±0 1±4 — 0±4 0±1 0±3 — 0±6 1±2 1±6 0±0 0±5 —
0 0±1 3 1 8 1 0 0 0 0 37 52 0 21 32 11 2 7 29 21 2 24 4
— 0±1 1±6 0±6 4±0 0±6 — — — — 7±0 1±6 — 3±6 1±9 1±2 2±0 0±5 4±1 2±3 1±7 3±1 2±6
664
AO, anti-oxidant (0±01 % butylated hydroxyanisole0±01 % butylated hydroxytoluene) ; DK, deodorised kerosene ; E, Edolex ; G, groundnut oil ; S, soya oil ; SS, Shellsol.
in germination over 10 % after 44 h incubation. These were sebacic and some acids of C12 and longer, but not lauric acid. There was little or no germination in the presence of acids previously shown to have a negative effect on germination, e.g. salicylic, caprylic, capric or eugenol. The majority of treatments showed some germination after 44 h incubation. Table 3 shows the effects of C12 (lauric), both alone and combined with stearic acids (C18), on the germination of conidia from long term storage samples. All but one sample, with the exception of one which had no germination (not shown in Table 4), increased their percentage germination with the addition of C12. Further increases were seen when stearic acid was added. In most cases there was about 10 % increase in germination after the addition of both C12 and stearic acids. Samples with low germination rates showed a proportional increase in germination. The results were analysed using a one-way ANOVA showing the least significant differences between the three formulations. The addition of acids had a significant effect on increasing germination (F , ¯ 6±7906 ; P ! 0±001). A Bonferroni # #(* modified Least Significant Differences Test showed that the addition of C12 increased germination and the further addition of stearic acid again increased it (both at P ! 0±5 %). However, this analysis minimises the true significance. Paired t-tests showed that the addition of C12 significantly improved germination compared with that of the control formulation (t ¯ 7±68, 99 .. P ! 0±001) as did the further addition of stearic acid compared with formulation plus C12 (t ¯ 8±57, 99 .. P ! 0±001). Strictly, paired t-tests should not be used for multiple comparisons. To compensate, the number of
S. E. Barnes and D. Moore
665
Table 4. Germination of conidia of Metarhizium flavoviride (%³..) in oil formulations with the addition of different amounts of capric acid and}or stearic acid (C18) Treatment Control
1 84³1±4
2 87³1±5
3 83³1±5
4 85³2
5 80³1±2
6 83³1±4
Ratio of formulations Control : capric
100 : 1 0
100 : 0±5 0
100 : 0±4 0
100 : 0±25 0
100 : 0±125 0
100 : 0±063 75³1±5
Control : stearic
100 : 1 89³1±2
100 : 0±5 88³0±7
100 : 0±4 88³0±5
100 : 0±25 89³1±06
100 : 0±125 84³0±85
100 : 0±063 84³0±9
Control : stearic : capric
100 : 1 : 1 0
100 : 1 : 0±5 4³1±5
100 : 1 : 0±37 49³3±63
100 : 1 : 0±25 79³1±6
100 : 1 : 0±125 79³0±8
100 : 1 : 0±063 85³1±0
comparisons made, three, can be multiplied by the level of significance achieved in these tests, still showing highly significant results (P ! 0±005). In the final experiment both a germination inhibitor, capric acid and a promoter, stearic acid (C18), were used (Table 4). Capric acid caused total inhibition of germination of conidia until a ratio of 1 ml of conidial formulation to 0±063 ml of 1 % capric was reached. The conidial formulation showed higher germination with the addition of stearic acid as with previous experiments. When both C10 and stearic acid were added a different pattern emerged. If the concentrations of the two fatty acids were equal there was no germination, but as the proportion of capric decreased there was an increase in the amount of germination. Germination first occurred when the proportion of capric was decreased to half that of stearic acid. At the final concentration (0±063 capric : 1 stearic acid), there was no inhibition of germination. D I S C U S S I ON This appears to be the first work carried out on the effects of various acids on germination of M. flavoviride and demonstrated both stimulation and inhibition of germination. Certain short chain fatty acids such as caprylic and capric completely inhibited germination. Sebacic and longer chained acids often showed stimulation, even in sample II which showed no control germination. We have no explanation why samples I and II, replicates from a standard procedure, should differ so widely in their germination. Loss of viability of M. flavoviride in storage is usually associated with excess moisture (Moore et al., 1966) ; a matter as simple as a defective seal may have allowed moisture into sample II. Another inconsistent result, which cannot be explained, was a stimulatory effect from the C12 sample which was not repeated by lauric acid (also C12) ; the two samples may have differed slightly but analysis by gas chromatography could not separate them (G. Saddler & C. Lowe, personal communication). The stimulatory effects caused by certain acids increased germination more when two were added to the same sample. This may be simply due to the increase in the concentration of the fatty acids or they may have a synergistic effect. Conidia may show poor viability because specific chemicals may become limiting and adding stearic acid, for example, overcomes this effect. There are practical implications from these laboratory
results which need detailed investigation. The insect cuticle provides the first barrier to infection by entomopathogenic fungi and inhibitory compounds may be very significant in host defence (Smith & Grula, 1982). The cuticle of Schistocerca gregaria contains the inhibitory capric acid in larger amounts than, for example, stearic acid, which had a stimulatory effect on the germination of M. flavoviride. In our work a larger proportion of stimulatory stearic acid (4–15 times as much) was required, in comparison to the quantity of capric acid, to produce a high percentage of germination. The use, as additives, of particular compounds to overcome inhibitory effects needs investigating. It has been shown that endogenous levels of stearic acid in fungi can be increased by manipulating the temperature and pH (Brown et al., 1990), and so it may be possible to induce sufficient levels of stimulatory fatty acids in conidia of M. flavoviride. It may also be possible to increase practical storage time of conidia by increasing endogenous levels or by using additives, before or after storage, to maintain viability. Reduced viability may be due, in part, to loss of important component acids. Even more intriguing is the possibility that certain combinations of fatty acids may protect an insect against specific fungal isolates and that it may be possible to overcome this resistance by adding a stimulatory fatty acid to a formulation. We would like to acknowledge the collaborative research programme on the biological control of locusts and grasshoppers (Lutte Biologique contre les Locustes et Sauteriaux, LUBILOSA) which is funded by the overseas aid agencies of Canada (CIDA), The Netherlands (DGIS), Switzerland (SDC) and the U.K. (ODA). We thank the LUBILOSA team for their support. Thanks are also due to David Coupland and Sue Pritchard (Croda Chemicals Ltd and John L. Seaton & Co Ltd) for assistance and supply of fatty acids, Gerry Saddler and Caroline Lowe (International Mycological Institute) for chemical analysis and Anja Rott for help with statistics. R E F E R E N C ES Bateman, R. P., Carey, M., Moore, D. & Prior, C. (1993). The enhanced infectivity of Metarhizium flavoviride in oil formulations to desert locusts at low humidities. Annals of Applied Biology 122, 145–152. Bidochka, M. J. & Khachatourians, G. G. (1992). Growth of the entomopathogenic fungus Beauveria bassiana on cuticular components from the migratory grasshopper, Melanoplus sanguinipes. Journal of Invertebrate Pathology 59, 165–173.
Effects of fatty acids on germination of conidia of M. flavoviride Boucias, D. G., Brasaemle, D. L. & Nation, J. L. (1984). Lipid composition of the entomopathogenic fungus Nomuraea rileyi. Journal of Invertebrate Pathology 43, 254–258. Brown, D. E., Hasan, M., Lepe-Casillas, M. & Thornton, A. J. (1990). Effect of temperature and pH on lipid accumulation by Trichoderma reesei. Applied Microbiology and Biotechnology 34, 335–339. Butt, T. M., Ibrahim, L., Clark, S. J. & Beckett, A. (1995). The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles. Mycological Research 99, 945–950. Kerwin, J. L. (1987). Fatty acids and fungal development : Structure–activity relationships. In Ecology and Metabolism of Plant Lipids (ed. G. Fuller & W. D. Nes), pp. 329–342. American Chemical Society, Symposium series No. 325, Washington, DC. Lecuona, R., Riba, G., Cassier P, & Clement, J. L. (1991). Alterations of insect epicuticular hydrocarbons during infection with Beauveria bassiana or B. brongniartii. Journal of Invertebrate Pathology 58, 10–18. Moore, D., Bateman, R. P., Carey, M. & Prior, C. (1995). Long-term storage of Metarhizium flavoviride conidia in oil formulations for the control of locusts and grasshoppers. Biocontrol Science and Technology 5, 193–199. (Accepted 17 October 1996)
666
Moore, D., Douro-Kpindou, O. K., Jenkins, N. E. & Lomer, C. J. (1966). Effects of moisture content and temperature on storage of Metarhizium flavoviride conidia. Biocontrol Science and Technology 6, 51–61. Oraha, V. S. & Lockey, K. H. (1990). Cuticular lipids of Locusta migratoria migratoriodes (R and F), Schistocerca gregaria (Forska/ l) (Acrididae) and other orthopteran species. I. Polar components. Comparative Biochemistry and Physiology 95 B, 603–608. Prior, C., Jollands, P. & le Patourel, G. (1988). Infectivity of oil and water formulations of Beauveria bassiana (Deuteromycotina : Hyphyomycetes) to the cocoa weevil pest Pantorhytes plutus (Coleoptera : Curculionidae). Journal of Invertebrate Pathology 52, 66–72. Ro$ bbelen, G., Downey, K. R. & Ashri, A. (1989). Oil Crops of the World, Their Breeding and Utilization. McGraw-Hill : New York. Smith, R. J. & Grula, E. A. (1982). Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana. Journal of Invertebrate Pathology 39, 15–22. Stathers, T. E., Moore, D. & Prior, C. (1993). The effect of different temperatures on the viability of Metarhizium flavoviride conidia stored in vegetable and mineral oils. Journal of Invertebrate Pathology 62, 111–115.
662
Printed in Great Britain
The effect of fatty, organic or phenolic acids on the germination of conidia of Metarhizium flavoviride
S. E. B A R N E S A N D D. M O O R E International Institute of Biological Control, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7TA, U.K.
Conidia of Metarhizium flavoviride were suspended in oil formulations and received fatty and other acids before germination. This was done by the acids being added either to the conidial suspensions before inoculation onto agar plates or directly into the agar before the conidia were applied. Certain acids of chain lengths C10 and below, including lactic, salicylic, caprylic and capric acids produced significant inhibition of germination when added to the oil formulations. When incorporated into the agar, all acids of C10 and below were inhibitory, with the exception of sebacic and succinic acids. Sebacic acid (a C10 saturated dicarboyxlic acid) and all the longer chain fatty acids sampled produced no inhibition of germination, either when added to the formulation or the agar. Tests were carried out on a sample of conidia which showed no germination ; a few acids stimulated some germination. These included sebacic acid and longer chain fatty acids such as stearic and (inconsistently) lauric acids. Conidia from a long term storage experiment showed increased germination on the addition of stearic and}or lauric acids. The inhibitory effect of capric acid on germination was overcome by a 4–15 times higher concentration of stearic acid.
The use of Metarhizium flavoviride for the biological control of locusts and grasshoppers is being researched in a collaborative project involving the International Institute of Biological Control (IIBC), the International Institute of Tropical Agriculture (IITA), Cotonou, in the Republic of Benin and the De! partement de Formation en Protection des Ve! ge! taux (DFPV), Niamey, Niger. The conidia of M. flavoviride are lipophilic and can be formulated in oil for application with ultra-low volume equipment. Formulation in oil increases efficacy and reduces dependence on humid conditions (Prior et al., 1988 ; Bateman et al., 1993) ; field trials have been successful in killing locusts and grasshoppers, but the speed of kill is slower than desired and research is being undertaken to reduce time to death. The formulation and storage of M. flavoviride conidia in oil is logistically easy. However, storage of conidia in different oils results in variable germination (Stathers et al., 1993 ; Moore et al., 1995) ; one factor causing this may be the presence of free fatty acids in the oils, some of which have been reported to inhibit, and others promote, germination of fungal conidia (Kerwin, 1987 ; Bidochka & Khachatourians, 1992). Most vegetable oils contain amongst others, palmitic, oleic and linoleic acids (Ro$ bbelen et al., 1989). Components of the cuticle of an insect may have both inhibitory and nutritional effects on the germination of conidia including, Beauveria bassiana and M. anisopliae ; these effects could be due, in part, to the fatty acids present on the cuticle (Lecuona et al., 1991; Butt et al., 1995). For Schistocerca gregaria (Forska/ l) these fatty acids occur at different levels, (C16 " C14 " C10 " C12 " C18) with 1±5 % of the cuticle’s
composition being free fatty acids for females and 4±7 % for males (Oraha & Lockey, 1990). Some fatty acids may stimulate the initiation of germination and encourage hyphal growth, whilst others may inhibit both these processes. Oleic and linoleic acids increase, whilst stearic and linolenic inhibit, mycelial growth of Beauveria bassiana (Bidochka and Khachatourians, 1992). Conidia of Nomuraea rileyi have been shown to rely completely on an exogenous supply of fatty acids and other substrates for germination (Boucias et al., 1984). Short chain fatty acids block the uptake of phosphate and thiamine in certain fungi, preventing the conidia from germinating (Kerwin, 1987). Fungi have fatty acid carrier systems which effect selective uptake through fungal membranes according to carbon chain length (Kerwin, 1987). This may influence which fatty acids inhibit and which promote germination. The major fungal fatty acids are unbranched C12 to C20 chains, mostly even numbered, the main examples in fungi being palmitoleic, oleic, linoleic and linolenic (Kerwin, 1987). In the present work we investigated effects of various fatty acids on the germination of conidia of M. flavoviride to determine if any means of hastening or increasing germination, and hence increasing the speed of kill, could be found. MATERIALS AND METHODS Fungal material An isolate of M. flavoviride (Metschn.) Sorokı- n(IMI 331089) had been obtained from Ornithacris cavroisi (Finot) (Orthoptera : Acrididae) at Niamey, Niger in 1988. Two replicate samples
S. E. Barnes and D. Moore of conidia (I & II), which had been dried to around 5 % moisture content and mixed with silica gel, were obtained after 10 months storage at 10 °C. One gram of each of these replicates was suspended in 200 ml of a 50}50 mix of two mineral oils used in pesticide applications, Shellsol, a light oil, and Ondina, a more viscous oil. The conidial suspensions (of approx. 2±5¬10) conidia ml−") were sonicated (Lucas Dawe Ultrasonicater Sonicleaner) for 3 min before being stored at 10° C. Other suspensions, of the same isolate, were taken from a long term experiment (Moore et al., 1995), the spores having been stored for 4 years at 8° in several oil mixtures. Fatty acids Twenty-two samples of various acids (Croda Chemicals Ltd, Goole, John L. Seaton and Co Ltd, Hull and Sigma Ltd, Poole) were tested for their effects on the germination of M. flavoviride. These included organic acids such as citric and salicylic, fatty acids, including dicarboxylic fatty acids such as succinic and a phenolic acid, eugenol. Only linoleic acid and eugenol were supplied by Sigma. Four fatty acids (C12, lauric ; C14, myristic : C16, palmitic and C18, stearic), came in two forms either named, or labelled by their carbon number ; these samples varied slightly in their commercial processing but were all of 95 % purity or better with impurities being of closely related acids. To distinguish the two forms of fatty acids specified above, samples labelled only by C numbers when supplied are referred by their C number followed by name eg C12 (lauric) in the text. Those named are referred to as, e.g., lauric (C12) in the text. Solutions of most of the fatty acids were initially made up to 2 % concentrations and added to an equal volume of oil formulation of conidia, to reduce the fatty acid concentration to 1 %. Some of the fatty acids were not soluble in the oil solutions at the higher concentrations ; in these cases a final concentration of 0±75 % fatty acid in the oil formulation was obtained (Table 1). In certain cases the oil was warmed to dissolve the acid. The control treatment was the conidial suspension with no added acids.
663 Table 1. Germination (mean percentage³CI) of Metarhizium flavoviride conidia in oil formulations. Fatty acids and other materials were either added to the oil formulation or incorporated into the agar (Sample I) Fatty acid added to formulation
Control Lactic Succinic Tartaric Adipic Citric Salicylic Caprylic Capric Eugenol Sebacic C12 (Lauric) Lauric C14 (Myristic) Myristic C16 (Palmitic) Palmitic C18 (Stearic) Stearic Linoleic Oleic Behenic Erucic
Fatty acid added to agar
% added
Mean
³95 % CI
Mean
³95 % CI
— 1 0±75 0±75 0±75 0±75 0±75 0±75 0±75 1 0±75 1 1 1 0±75 1 0±75 1 0±75 1 1 0±04 1
84 5 84 87 84 61 16 0 0 3 85 87 81 86 83 83 86 86 86 89 89 85 83
8±8 13±4 7±3 5±1 8±1 38±9 9±0 — — 5±5 8±1 7±7 19±9 3±7 18±9 12±9 6±0 0±6 3±1 1±1 4±8 4±2 10±6
88 0±4 86 35 27 66 0 0 0 0 65 73 86 89 86 91 88 89 79 NA 89 89 81
4±2 1±0 11±2 17±0 24±8 20±0 — — — — 38±0 27±8 4±1 3±9 7±5 4±3 9±2 4±5 13±4 NA 5±9 4±8 35±0
NA, not available. It was not possible to obtain a count from these plates as the agar was clouded ; growth was seen on the plates after several days.
for each treatment of suspension I at 24 h and from three for sample II at 24 and 44 h. Experiment 2 : Germination counts on plates containing fatty acids Acids were added to molten SDA, and mixed before been poured onto plates which were inoculated with conidial suspension I. Germination counts were carried out on five plates per treatment assessed after 24 h at 25°.
Viability test The percentage germination was determined by spreading 0±1 ml of each oil suspension on SDA plates (Sabouraud Dextrose Agar) (Oxoid, Basingstoke) with a small spatula. The plates were incubated at 25°, usually for 24 h but for 44 h where conidia were slower to germinate. The plates were scanned at ¬300 magnification, and at least 300 conidia were assessed on each plate. Single conidia were assessed to avoid difficulties in determining the source of germ tubes in clumps of conidia. Germination was considered to have occurred when the germ tubes projected from the conidia to a length approx half that of the length of the conidia. Experiment 1 : Percentage germination after inoculation with fatty acids Acids were added to conidial suspensions I and II as outlined above. Germination counts were carried out from five plates
Experiment 3 : Fatty acids on previously stored samples Germination counts were carried out on 21 samples of M. flavoviride in a number of oils from long term storage experiments (Moore et al., 1995). Stearic acid (C18) at 0±5 % and C12 (lauric) at 1 % concentration were added to equal volumes of conidial formulating resulting in 0±25 and 0±5 % concentrations respectively. Five plates were assessed for germination from each treatment after 24 h at 25°. Experiment 4 : Tests using both an inhibitory and a stimulatory fatty acid Suspension I was combined with both capric acid, which had been shown to inhibit germination, and stearic acid (C18), either separately or together. The treatments started as a 1 % concentration of capric and}or stearic acid in an oil formulation of conidia. The combined treatments of formulation : stearic :
Effects of fatty acids on germination of conidia of M. flavoviride capric began at the ratio of 100 : 1 : 1. Capric acid concentrations were decreased in subsequent treatments, down to a 100 : 1 : 0±063 ratio. Five replications were carried out for each treatment.
Table 3. Germination of Metarhizium flavoviride conidia stored in a range of oils for 4 years with or without added C12 (lauric) and}or stearic (C18) fatty acids
Formulation
Formulation C12
Formulation C12 stearic
Oil used
Mean (%)
³..
Mean (%)
³..
Mean (%)
³..
GSSAO SSSAO ESS GDK GSS SDKAO GDKAO EDK SDK SSS GSSAO SSSAO ESS GDK GSS SDKAO GDKAO EDK SDK SSS
52 71 81 83 80 16 74 87 74 62 65 78 85 89 90 52 81 85 75 75
3±0 3±6 2±3 2±4 2±6 1±1 1±5 2±5 2±6 4±4 2±2 2±9 1±7 1±1 0±7 6±1 1±6 3±5 2±8 4±5
56 74 85 88 85 18 83 90 80 67 75 77 86 92 91 59 89 89 79 80
3±8 2±9 1±4 1±5 3±5 2±4 2±7 1±5 1±6 2±7 1±4 4±9 1±1 0±4 1±4 2±9 0±8 1±7 3±4 2±6
66 80 88 92 91 22 83 93 86 76 77 84 90 92 95 67 91 92 85 87
3±0 4±0 2±5 1±8 1±6 1±8 1±4 1±2 3±1 4±3 2±9 1±9 0±5 0±8 0±7 4±5 0±7 0±5 1±6 1±9
RESULTS The results of treatments to sample I in experiment 1 (Table 1), showed that the addition of most fatty acids did not reduce germination compared with the control level of 84±1 %. However, caprylic and capric acids, when added to the oil formulation, completely inhibited germination, while additionally eugenol and salicylic were completely inhibitory when included in the agar treatment. Other treatments also caused marked inhibition of germination when added to the oil formulation or agar plates. Citric, sebacic and C12 (lauric) acids may have been mildly inhibitory as replicates results were variable, indicated by high confidence intervals. Little or no effect on percentage germination was demonstrated by succinic, sebacic (added to oil) and C12 or longer chain acids. The high germination rate of the control did not lend itself to showing substances which promote germination. There was no significant difference in the effects on germination caused by the fatty acids in the two different treatment methods, oil and agar, except for adipic (F , ¯ 36±59 ; P ! 0±001), salicylic ") (F , ¯ 6±05 ; P ! 0±05) and tartaric acids (F , ¯ 67±28 ; P ! ") ") 0±001). In each case germination on agar plus acid was significantly lower than that of conidia in oil plus acid. In contrast, sample II showed no control germination, but the addition of some acids stimulated germination (Table 2), although usually at low levels. Only eight treatments resulted Table 2. Germination of Metarhizium flavoviride conidia in oil formulation after adding fatty acids and other materials and incubating for 24 or 44 h (Sample II) 24 h
Control Lactic Succinic Tartaric Adipic Citric Salicylic Caprylic Capric Eugenol Sebacic C12 (Lauric) Lauric C14 (Myristic) Myristic C16 (Palmitic) Palmitic C18 (Stearic) Stearic Linoleic Oleic Behenic Erucic
44 h
Mean (%)
³..
Mean (%)
³..
0 0 0±3 0 1 0±1 1 0 0 0 13 10 0 1 5 0±3 0 1 2 3 0 1 0
— — 0±3 0±0 1±1 0±1 0±5 — — — 3±0 1±4 — 0±4 0±1 0±3 — 0±6 1±2 1±6 0±0 0±5 —
0 0±1 3 1 8 1 0 0 0 0 37 52 0 21 32 11 2 7 29 21 2 24 4
— 0±1 1±6 0±6 4±0 0±6 — — — — 7±0 1±6 — 3±6 1±9 1±2 2±0 0±5 4±1 2±3 1±7 3±1 2±6
664
AO, anti-oxidant (0±01 % butylated hydroxyanisole0±01 % butylated hydroxytoluene) ; DK, deodorised kerosene ; E, Edolex ; G, groundnut oil ; S, soya oil ; SS, Shellsol.
in germination over 10 % after 44 h incubation. These were sebacic and some acids of C12 and longer, but not lauric acid. There was little or no germination in the presence of acids previously shown to have a negative effect on germination, e.g. salicylic, caprylic, capric or eugenol. The majority of treatments showed some germination after 44 h incubation. Table 3 shows the effects of C12 (lauric), both alone and combined with stearic acids (C18), on the germination of conidia from long term storage samples. All but one sample, with the exception of one which had no germination (not shown in Table 4), increased their percentage germination with the addition of C12. Further increases were seen when stearic acid was added. In most cases there was about 10 % increase in germination after the addition of both C12 and stearic acids. Samples with low germination rates showed a proportional increase in germination. The results were analysed using a one-way ANOVA showing the least significant differences between the three formulations. The addition of acids had a significant effect on increasing germination (F , ¯ 6±7906 ; P ! 0±001). A Bonferroni # #(* modified Least Significant Differences Test showed that the addition of C12 increased germination and the further addition of stearic acid again increased it (both at P ! 0±5 %). However, this analysis minimises the true significance. Paired t-tests showed that the addition of C12 significantly improved germination compared with that of the control formulation (t ¯ 7±68, 99 .. P ! 0±001) as did the further addition of stearic acid compared with formulation plus C12 (t ¯ 8±57, 99 .. P ! 0±001). Strictly, paired t-tests should not be used for multiple comparisons. To compensate, the number of
S. E. Barnes and D. Moore
665
Table 4. Germination of conidia of Metarhizium flavoviride (%³..) in oil formulations with the addition of different amounts of capric acid and}or stearic acid (C18) Treatment Control
1 84³1±4
2 87³1±5
3 83³1±5
4 85³2
5 80³1±2
6 83³1±4
Ratio of formulations Control : capric
100 : 1 0
100 : 0±5 0
100 : 0±4 0
100 : 0±25 0
100 : 0±125 0
100 : 0±063 75³1±5
Control : stearic
100 : 1 89³1±2
100 : 0±5 88³0±7
100 : 0±4 88³0±5
100 : 0±25 89³1±06
100 : 0±125 84³0±85
100 : 0±063 84³0±9
Control : stearic : capric
100 : 1 : 1 0
100 : 1 : 0±5 4³1±5
100 : 1 : 0±37 49³3±63
100 : 1 : 0±25 79³1±6
100 : 1 : 0±125 79³0±8
100 : 1 : 0±063 85³1±0
comparisons made, three, can be multiplied by the level of significance achieved in these tests, still showing highly significant results (P ! 0±005). In the final experiment both a germination inhibitor, capric acid and a promoter, stearic acid (C18), were used (Table 4). Capric acid caused total inhibition of germination of conidia until a ratio of 1 ml of conidial formulation to 0±063 ml of 1 % capric was reached. The conidial formulation showed higher germination with the addition of stearic acid as with previous experiments. When both C10 and stearic acid were added a different pattern emerged. If the concentrations of the two fatty acids were equal there was no germination, but as the proportion of capric decreased there was an increase in the amount of germination. Germination first occurred when the proportion of capric was decreased to half that of stearic acid. At the final concentration (0±063 capric : 1 stearic acid), there was no inhibition of germination. D I S C U S S I ON This appears to be the first work carried out on the effects of various acids on germination of M. flavoviride and demonstrated both stimulation and inhibition of germination. Certain short chain fatty acids such as caprylic and capric completely inhibited germination. Sebacic and longer chained acids often showed stimulation, even in sample II which showed no control germination. We have no explanation why samples I and II, replicates from a standard procedure, should differ so widely in their germination. Loss of viability of M. flavoviride in storage is usually associated with excess moisture (Moore et al., 1966) ; a matter as simple as a defective seal may have allowed moisture into sample II. Another inconsistent result, which cannot be explained, was a stimulatory effect from the C12 sample which was not repeated by lauric acid (also C12) ; the two samples may have differed slightly but analysis by gas chromatography could not separate them (G. Saddler & C. Lowe, personal communication). The stimulatory effects caused by certain acids increased germination more when two were added to the same sample. This may be simply due to the increase in the concentration of the fatty acids or they may have a synergistic effect. Conidia may show poor viability because specific chemicals may become limiting and adding stearic acid, for example, overcomes this effect. There are practical implications from these laboratory
results which need detailed investigation. The insect cuticle provides the first barrier to infection by entomopathogenic fungi and inhibitory compounds may be very significant in host defence (Smith & Grula, 1982). The cuticle of Schistocerca gregaria contains the inhibitory capric acid in larger amounts than, for example, stearic acid, which had a stimulatory effect on the germination of M. flavoviride. In our work a larger proportion of stimulatory stearic acid (4–15 times as much) was required, in comparison to the quantity of capric acid, to produce a high percentage of germination. The use, as additives, of particular compounds to overcome inhibitory effects needs investigating. It has been shown that endogenous levels of stearic acid in fungi can be increased by manipulating the temperature and pH (Brown et al., 1990), and so it may be possible to induce sufficient levels of stimulatory fatty acids in conidia of M. flavoviride. It may also be possible to increase practical storage time of conidia by increasing endogenous levels or by using additives, before or after storage, to maintain viability. Reduced viability may be due, in part, to loss of important component acids. Even more intriguing is the possibility that certain combinations of fatty acids may protect an insect against specific fungal isolates and that it may be possible to overcome this resistance by adding a stimulatory fatty acid to a formulation. We would like to acknowledge the collaborative research programme on the biological control of locusts and grasshoppers (Lutte Biologique contre les Locustes et Sauteriaux, LUBILOSA) which is funded by the overseas aid agencies of Canada (CIDA), The Netherlands (DGIS), Switzerland (SDC) and the U.K. (ODA). We thank the LUBILOSA team for their support. Thanks are also due to David Coupland and Sue Pritchard (Croda Chemicals Ltd and John L. Seaton & Co Ltd) for assistance and supply of fatty acids, Gerry Saddler and Caroline Lowe (International Mycological Institute) for chemical analysis and Anja Rott for help with statistics. R E F E R E N C ES Bateman, R. P., Carey, M., Moore, D. & Prior, C. (1993). The enhanced infectivity of Metarhizium flavoviride in oil formulations to desert locusts at low humidities. Annals of Applied Biology 122, 145–152. Bidochka, M. J. & Khachatourians, G. G. (1992). Growth of the entomopathogenic fungus Beauveria bassiana on cuticular components from the migratory grasshopper, Melanoplus sanguinipes. Journal of Invertebrate Pathology 59, 165–173.
Effects of fatty acids on germination of conidia of M. flavoviride Boucias, D. G., Brasaemle, D. L. & Nation, J. L. (1984). Lipid composition of the entomopathogenic fungus Nomuraea rileyi. Journal of Invertebrate Pathology 43, 254–258. Brown, D. E., Hasan, M., Lepe-Casillas, M. & Thornton, A. J. (1990). Effect of temperature and pH on lipid accumulation by Trichoderma reesei. Applied Microbiology and Biotechnology 34, 335–339. Butt, T. M., Ibrahim, L., Clark, S. J. & Beckett, A. (1995). The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles. Mycological Research 99, 945–950. Kerwin, J. L. (1987). Fatty acids and fungal development : Structure–activity relationships. In Ecology and Metabolism of Plant Lipids (ed. G. Fuller & W. D. Nes), pp. 329–342. American Chemical Society, Symposium series No. 325, Washington, DC. Lecuona, R., Riba, G., Cassier P, & Clement, J. L. (1991). Alterations of insect epicuticular hydrocarbons during infection with Beauveria bassiana or B. brongniartii. Journal of Invertebrate Pathology 58, 10–18. Moore, D., Bateman, R. P., Carey, M. & Prior, C. (1995). Long-term storage of Metarhizium flavoviride conidia in oil formulations for the control of locusts and grasshoppers. Biocontrol Science and Technology 5, 193–199. (Accepted 17 October 1996)
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Moore, D., Douro-Kpindou, O. K., Jenkins, N. E. & Lomer, C. J. (1966). Effects of moisture content and temperature on storage of Metarhizium flavoviride conidia. Biocontrol Science and Technology 6, 51–61. Oraha, V. S. & Lockey, K. H. (1990). Cuticular lipids of Locusta migratoria migratoriodes (R and F), Schistocerca gregaria (Forska/ l) (Acrididae) and other orthopteran species. I. Polar components. Comparative Biochemistry and Physiology 95 B, 603–608. Prior, C., Jollands, P. & le Patourel, G. (1988). Infectivity of oil and water formulations of Beauveria bassiana (Deuteromycotina : Hyphyomycetes) to the cocoa weevil pest Pantorhytes plutus (Coleoptera : Curculionidae). Journal of Invertebrate Pathology 52, 66–72. Ro$ bbelen, G., Downey, K. R. & Ashri, A. (1989). Oil Crops of the World, Their Breeding and Utilization. McGraw-Hill : New York. Smith, R. J. & Grula, E. A. (1982). Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana. Journal of Invertebrate Pathology 39, 15–22. Stathers, T. E., Moore, D. & Prior, C. (1993). The effect of different temperatures on the viability of Metarhizium flavoviride conidia stored in vegetable and mineral oils. Journal of Invertebrate Pathology 62, 111–115.