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Aerodynamic diameter of conidia of Erynia neoaphidis and other entomophthoralean fungi
Mycol. Res. 106 (2) : 233–238 (February 2002).
# The British Mycological Society
233
DOI : 10.1017\S0953756201005275 Printed in the United Kingdom.
Aerodynamic diameter of conidia of Erynia neoaphidis and other entomophthoralean fungi
Farhad HEMMATI1,3†, Judith K. PELL1*, H. Alastair McCARTNEY2 and Michael L. DEADMAN3‡ " Plant and Invertebrate Ecology Division, # Plant Pathogen Interactions Division, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK. $ Department of Agriculture, University of Reading, Reading RG6 6AT, UK. E-mail : judith.pell!bbsrc.ac.uk Received 5 March 2001 ; accepted 16 September 2001.
The aerodynamic diameter, da, of conidia produced in vivo and in vitro by the entomopathogenic fungus Erynia neoaphidis were estimated using an impaction method. The estimated values of da for conidia produced in vivo were smaller than those produced in vitro : in vivo the values of da for primary and secondary conidia were between 16 and 18 µm (equivalent fall speed, Vs, 0n8–1n0 cm s−") ; for in vitro produced conidia da values were between 28 and 31 µm (Vs, 2n4–3 cm s−"). For conidia produced by field collected cadavers the value of da, was estimated to be similar to that for conidia produced in vivo in the laboratory. The aerodynamic diameter of primary conidia of Conidiobolus obscurus (strain X39) and Zoophthora radicans (strain NW250) produced in vivo were also measured using the same method. The values of da for these two species were 45 and 17 µm (Vs, 6n2 and 0n9 cm s−") respectively. Implications for dispersal of E. neoaphidis are discussed. The physical diameters of the test spores were measured microscopically and compared with the aerodynamic diameters.
INTRODUCTION Entomopathogenic fungi, such as Erynia neoaphidis, are potentially useful biocontrol agents against aphids. However, their effectiveness relies on the early establishment of epizootics within the target insect population. The development of epizootics depends on the ability of the pathogen to spread in time and space. E. neoaphidis infects and kills a number of species of aphid, and spreads by discharging conidia from dead aphids into the air (Hemmati et al. 2001). The conidia are dispersed by wind to other aphid populations where they can initiate infection. The efficiency of dispersal of fungal spores (including those of entomopathogenic fungi), especially within crops, depends on the airflow and on the aerodynamic properties of the spores. The aerodynamic properties of spores determine the rate at which they fall from the air (fall speed) and the rate they are deposited onto surfaces by settling and by impaction (McCartney & Fitt 1985). In most models describing spore dispersal the aerodynamic characteristics of the spores are defined using one parameter * Corresponding author. † Current address : AREEO, Scientific & Research Bureau, PO Box 19835-111, Tehran, Iran. ‡ Current address : Department of Crop Sciences, Sultan Qaboos University, PO Box 34, Al Khod 123, Sultanate of Oman.
called the aerodynamic diameter, da (Legg & Powell 1975, Aylor & Taylor 1983) which is defined as the diameter of a unit density (1 g cm−$) sphere that has the same fall speed, Vs as the spore (May 1975, Chamberlain 1975). The fall speed is also referred to as the settling velocity or terminal velocity and is the speed at which the spore falls through still air when drag and gravity forces balance. The parameter da is used to calculate the rate of deposition of spores onto surfaces by impaction. The fall speed, and hence aerodynamic diameter can be calculated for particles of simple shape, such as spheres or cylinders, if the density of the particle is known (Chamberlain 1975). However, fungal spores rarely have such simple shapes, and even for nearly spherical spores da is often less than the diameter, d (Gregory 1973, McCartney 1991). E. neoaphidis actively discharges conidia (primary conidia) from aphid cadavers. After deposition primary conidia can actively discharge smaller, secondary conidia. Primary conidia are lemon-shaped, and secondary conidia are either of similar shape or more rounded (Morgan et al. 1995). The size range of primary conidia has been reported to be between 18–35 µm long and 11–15 µm wide and the range of the length\diam ratio (L\D) as 1n7–2n3. For secondary conidia, the equivalent ranges are : length 14–24 µm, width 11–20 µm and L\D 1n1–1n4 (Remaudie' re & Hennebert 1980, Wilding & Brady
Aerodynamics of entomophthoralean conidia 1984, Brown 1985). Morgan (1994) reported that the size of primary conidia (isolate X4) produced in vitro were significantly larger (mean 32i15n4 µm) than those produced in vivo (mean 19n8i11n8 µm). Eilenberg, Bresciani & Latge! (1990) showed that primary conidia of Entomophthora schizophorae produced in vitro were larger than those produced in vivo. As far as we are aware, there are no reports of values of Vs for E. neoaphidis conidia. Furthermore Vs has been measured for spores of only a few entomopathogenic fungi. For example, Sawyer, Griggs & Wayne (1994) measured Vs of Zoophthora radicans (isolates ARSEF 2282 and 2283), Conidiobolus thromboides (ARSEF 40) and C. obscurus (ARSEF 70) and found mean values of 0n36, 0n37, 1n39 and 4n49 cm s−", respectively. The results of measurements of the aerodynamic diameter of primary and secondary conidia of E. neoaphidis, which were produced both in vivo and in vitro, are reported. Measurements of da and Vs of in vivo-produced conidia of Conidiobolus obscurus and Zoophthora radicans are also presented. MATERIALS AND METHODS Conidia sources Sources of in vivo conidia Two sources of Erynia neoaphidis conidia were used. One was cadavers of pea aphids, Acyrthosiphon pisum, infected in the laboratory using the methods of Wilding (1973) with an isolate of E. neoaphidis (S1 ; from the Rothamsted culture collection). This isolate was originally collected from the nettle aphid, Microlophium carnosum on the Rothamsted farm. The other source was E. neoaphidis-infected, dried cadavers of rose-grain aphids, Metopolophium dirhodum, collected from naturally occurring epizootics in wheat fields on Rothamsted farm. Acyrthosiphon pisum aphids were infected with Conidiobolus obscurus (X39 from the Rothamsted culture collection) and diamondback moths, Plutella xylostella, were infected with Zoophthora radicans (reference NW 250 R (Malaysia) from the Rothamsted culture collection) in the laboratory. Infected cadavers of each insect species were used as sources of conidia for these species of fungi. Conidia sources for each experiment consisted of ten sporulating cadavers of the test species. The infected cadavers were placed on the base of a 50 ml, 2n5 cm diam, centrifuge tube lined with wet tissue paper. The tube was incubated at 10 mC and 100 % RH in darkness overnight to encourage sporulation. Source of in vitro conidia Erynia neoaphidis (S1) was grown on Sabouraud dextrose agar supplemented with milk and egg yolk (Wilding & Brobyn 1980) at 20 m in darkness for 3–4 wk prior to experimentation. Sections of mycelia cut from the growing edge of the colony were placed inside the
234 centrifuge tubes and incubated at 10 m, 100 % RH in darkness for 12 h, to encourage sporulation. Secondary conidia were produced from primary conidia from both in vitro and in vivo sources in the same way. Primary conidia were collected for 6 h in Petri dishes containing 1 % water agar, at 20 m and 100 % RH. A section of water-agar containing primary conidia was then placed in a 50 ml centrifuge tube. The primary conidia sporulated on the water agar providing a source of secondary conidia. The impaction method for estimating aerodynamic diameter (see below) was also tested using Lycopodium clavatum and Calvatia gigantea (giant puff-ball) spores. Lycopodium clavatum spores were obtained, as dry spores, from BDH Laboratory Chemicals (Poole, UK) and C. gigantea spores were obtained from a mature giant puff-ball collected on Rothamsted farm. Estimation of the aerodynamic diameter and settling velocity of conidia The aerodynamic diameter of test conidia was estimated from their impaction characteristics using the method described by McCartney, Schmechel & Lacey (1993). This method uses a May Ultimate Impactor (MUI, Research Engineers, London ; May 1975). The MUI is a cascade impactor used to classify particles according to their aerodynamic characteristics. Air is drawn through a series of seven stages, each consisting of a jet formed by a slit held above a removable microscope slide. Each stage collects smaller particles than its predecessor. For a uniformly sized sample, the proportion, P, of the total sample deposited on each stage depends only on the aerodynamic diameter of the particles (McCartney et al. 1993). For the first stage, P increases monotonically with aerodynamic size of the particles, but for the other stages, P increases to a maximum value then declines. The mean aerodynamic diameter of a sample can be estimated if the values of P for each stage are measured (McCartney et al. 1993). For each experiment, conidia from the test source were introduced into the MUI by inverting the centrifuge tube containing the source above the inlet : sporulating cadavers (primary conidia), sporulating mycelia (primary conidia), or sporulating primary conidia (secondary conidia). Nylon netting placed across the mouth of the centrifuge tube ensured that the source remained inside the tube and only the conidia passed into the MUI. The MUI was operated for 3 h with sources producing primary conidia and 15 h for sources producing secondary conidia. Microscope slides used in the MUI were coated with a layer of a mixture of petroleum jelly (Vaseline) and paraffin wax (100 : 18 w\w ; British Aerobiology Federation 1996) to retain conidia. After exposure, slides were removed from the MUI and the numbers of conidia collected on five identical traverses across the whole width of each slide were counted. One traverse was across the centre of the trace, the others were 1 and
F. Hemmati and others
235
2 cm on either side of the central traverse. The traverse width was 1 mm, the magnification i100 and the distance between the traverses 5 mm. The counts were used to estimate the total number of conidia deposited in the MUI and the proportion, P, collected on each stage. The average value of the aerodynamic diameter, da for sampled conidia in each test was estimated from the measured values of P for each stage using the methods described in McCartney et al. (1993). Briefly, the mean value of da was taken as the value that minimised the chi-squared value of P (i.e. (PmeasuredkPexpected)#\ Pexpected) when summed over all the stages which collected conidia. Pmeasured were the values of P measured for each stage, and Pexpected were the theoretical values for a monodisperse sample of aerodynamic diameter da (McCartney et al. 1993). Settling velocity (Vs) was determined from Chamberlain (1975) : (1) Vs l 0n00308 d#a where da is in µm and Vs is in cm s−". All experiments were done at 20 mC and two trials (replicates) were made for each conidium type, except for Erynia neoaphidis when two additional trials were done with laboratory produced cadavers (in vivo). Further trials were done using Lycopodium clavatum and Calvatia gigantea spores. The aerodynamic properties of these spores are known and the tests were done as an additional check on the method. The maximum width and length of conidia of each entomopathogen was estimated by measuring 50 conidia from each treatment using a micrometer graticule at i500 magnification. Condia were selected randomly by moving the stage across the slide and selecting conidia from the centre of the field from those caught on all stages of the MUI. RESULTS There was some variation in the size and shape of
conidia for the three entomopathogenic species tested (Table 1). Both primary and secondary conidia derived from E. neoaphidis (S1) cultured in vitro were longer and wider, but had a shorter length\width ratio, than in vivo conidia derived from pea aphid cadavers (Table 1). The mean values of length (L ), width (W ) and L\W were statistically different (t-test) for the three species. The mean length and width of primary conidia produced from field collected cadavers was significantly larger (P 0n05) than that of those produced in vivo in the laboratory, but significantly shorter (P 0n05) than those produced in vitro. However, E. neoaphidis conidia produced by Metopolophium dirhodum cadavers collected in the field, tended to be more elongated than the others (mean L\W values were 1n98, 1n89 and 1n67 for field in vivo, laboratory in vivo and laboratory in vitro respectively, and all were significantly different to each other, P 0n05). Secondary conidia tended to be smaller than primary conidia, except that the widths of primary and secondary conidia produced by the S1 strain in vivo were not significantly different (P l 0n5). Primary conidia of Zoophthora radicans produced from cadavers of Plutella xylostella tended to be shorter and narrower, but more elongated than E. neoaphidis conidia, while primary conidia of Conidiobolus obscurus, produced from Acyrthosiphon pisum, were larger, but more spherical (Table 1). In all experiments, most conidia were collected on stages 1–4 of the impactor (Table 2). Conidia of C. obscurus were found mostly on stages 1 and 2. In all experiments, the estimated aerodynamic diameter of conidia was greater than their mean maximum width and length, except for the primary conidia of fieldcollected E. neoaphidis in which the aerodynamic diameter was larger than the conidia’s width, but was smaller than their length (Table 3). Primary conidia of C. obscurus had the largest estimated value of da and in vivo produced conidia of E. neoaphidis had the smallest. Both primary and secondary conidia of E. neoaphidis (S1) cultured in vitro had larger estimated aerodynamic diameters than those cultured in vivo (Table 3).
Table 1. Dimensions of conidia of test fungi. Conidia-source Erynia neoaphidis (S1), in vivo, primary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), in vivo, primary Zoophthora radicans (NW250), in vivo, primary
Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range
Length (µm)
Width (µm)
Length\width ratio
18n8 (2n9) 13–25n9 16n5 (2n3) 11n1–22n2 26n7 (2n3) 22n2–31n5 21n6 (2n8) 14n8–27n8 23n5 (2n2) 18n5–29n6 33n4 (2n3) 28n9–40n0 15n9 (2n2) 11n1–20n6
10n2 (1n6) 7n4–16n7 10n4 (1n6) 7n4–14n8 16n2 (2n1) 13n0–20n6 17n1 (1n7) 13n0–20n6 12n0 (1n3) 9n3–14n8 27n8 (2n0) 23n3–32n2 7n3 (1n1) 5n6–11n1
1n89 (0n39) 1n11–2n78 1n61 (0n23) 1n19–2n01 1n67 (0n21) 1n30–2n17 1n27 (0n16) 1n0–1n89 1n98 (0n27) 1n50–2n50 1n21 (0n06) 1n06–1n33 2n20 (0n38) 1n49–3n68
Aerodynamics of entomophthoralean conidia
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Table 2. Percentage of conidia collected in each stage of the May Ultimate Impactor. Stage no.
Species (isolate in parenthesis)
Trial
Total number of conidia counted
1
2
3
4
5
6
7
Erynia neoaphidis (S1), in vivo, primary E. neoaphidis" (S1), in vivo, primary & secondary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), in vivo, primary Zoophthora radicans (NW250), in vivo, primary
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
42 971 29 360 6884 1129 6106 5455 15 562 13 265 19 668 4294 9273 11 610 1461 2576 8014 14 974
8n56 10n69 2n96 5n31 3n21 8n05 24n70 32n59 25n41 30n79 10n50 10n79 94n46 92n97 2n35 2n86
31n10 27n02 42n18 71n80 70n29 59n82 35n36 34n38 32n74 47n74 49n29 40n72 5n41 5n36 44n75 41n83
39n43 43n41 36n7 17n98 23n58 30n63 32n52 28n90 34n40 21n15 34n32 42n33 0n14 1n67 40n40 41n12
16n40 13n75 13n49 4n87 2n01 1n17 3n53 3n05 6n34 0n30 3n56 2n43 0 0 11n21 13n26
3n59 4n07 4n31 0 0n72 0n16 0n76 0n96 0n94 0n02 1n23 1n90 0 0 0n80 0n77
0n84 0n77 0n33 0 0n18 0n16 0n13 0n13 0n18 0 1n05 1n68 0 0 0n37 0n13
0n07 0n28 0 0 0 0 0 0 0 0 0n05 0n14 0 0 0n12 0n03
" The same source was used for each trial and the trials were done consecutively. No discrimination was made between primary and secondary conidia ; all conidia were counted.
Table 3. Aerodynamic characteristics of conidia estimated from the deposit on the May Ultimate Impactor. Aerodynamic diameter, da and fall speed, Vs. Trial 1
Trial 2 χ#
Conidia source
Mean LiW (µm)
da (µm)
da (µm)
Erynia neoaphidis (S1), in vivo, primary E. neoaphidis" (S1), in vivo, primary & secondary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), primary Zoophthora radicans (NW250), primary
18n8i10n2 —
16 17
6n5 8n7
16 18
16n5i10n4 26n7i16n2 21n6i17n1 23n5i12 33n4i27n8 15n9i7n3
18 29 30 17 45 17
9n6 15n9 17n5 0n4 0n01 9n3
17 31 28 17 45 17
Trial 1 Vs (cm s−")
Trial 2 Vs (cm s−")
4n2 5n8
0n8 0n9
0n8 1n0
3n3 10n9 4n6 0n5 0n03 9n7
1n0 2n6 2n8 0n9 6n2 0n9
0n9 3n0 2n4 0n9 6n2 0n9
χ#
" Experiments were made consecutively using the same source ; all conidia were counted.
DISCUSSION Experiments were done with two in vivo sources of Erynia neoaphidis, one cultured in the laboratory on pea aphids (Acyrthosiphon pisum) and one derived from field collected cadavers of rose grain aphids (Metopolophium dirhodum). Although the conidia derived from field collected cadavers were larger and more elongated than those produced from pea aphids, the estimated values of da were similar. This suggests that dispersal characteristics for these conidia would also be similar. The aerodynamic diameters of E. neoaphidis conidia were closer in size to the length of the conidia than to their width. For non-spherical particles, aerodynamic diameters are expected to be smaller than the largest dimension, but larger than the smaller dimension. For example, for elongated conidia of Alternaria species, da was found to range between about 10 and 40 µm while the length varied between about 40 and 150 µm
(McCartney et al. 1993). Aerodynamic diameters are defined as the diameter of a unit density (1 g cm−$) sphere with the same aerodynamic characteristics. Thus the values of da estimated for E. neoaphidis conidia would suggest that their densities were greater than that of water. Alternatively, as da values were estimated using impaction, the uneven distribution of mass within the conidia combined with its shape may have enhanced the impaction efficiency of the conidia by orientating the conidia to reduce drag, this would have also increased the estimate of da. The results of additional tests using Lycopodium clavatum and C. gigantea spores to check the impaction method, suggested that the impaction method did not overestimate da values. Estimates of da for L. clavatum spores were between 25 and 27 µm, slightly less than the measured diameter of about 30 µm, and about 5 µm for 4n5 µm diameter C. gigantea spores. It therefore seems unlikely that the impaction method significantly overestimated the aerodynamic diameter of the E. neoaphidis conidia.
F. Hemmati and others The aerodynamic diameters of Conidiobolus obscurus and Zoophthora radicans estimated in these experiments were larger than their physical dimensions. This also suggests that the densities of these spores were greater than that of water or that their shape or mass distribution enhanced their impaction efficiencies. Conidia of both species are not spherical. The fall speeds corresponding to estimated da were larger than those measured by Sawyer et al. (1994). They measured fall speed by direct observation of conidia falling in narrow glass tubes and noted that the conidia of C. obscurus, in particular, fell more slowly than predicted by Stokes’ law. Their results may have been influenced by either wall (Edmonds 1979) or electrostatic effects. With the exception of species of Massospora, all entomophthoralean fungi produce actively discharged conidia, which is clearly an adaptation for dispersal by wind (Hemmati et al. 2001, Pell et al. 2001). An understanding of the wind-borne dispersal of such fungi may provide an insight into how they have evolved to exploit different ecological niches within the agro-environment. For example, for the two aphid pathogens E. neoaphidis and C. obscurus, da for E. neoaphidis is smaller than that for C. obscurus and so it is better adapted to aerial dispersal. C. obscurus produces resting spores for survival in the absence of the host and these are not known in E. neoaphidis. This suggests that even if E. neoaphidis causes local extinctions in its host population it is able to disperse to other host populations. An understanding of spore dispersal capabilities of entomopathogenic fungi is also useful in assessing their suitability in different biological control strategies. For example, if the target pest is patchily distributed or alternate hosts are being exploited as reservoirs of a specific fungus then it is essential that the fungus can disperse between those populations, particularly between reservoir and target populations. However, if a fungus is to be applied as a mycoinsecticide to an area of crop, with the goal of initiating a large epizootic rapidly in the local area, then a species which is deposited rapidly would be more effective. Thus, E. neoaphidis may be more suited to the first type of strategy than C. obscurus : because of its smaller da it would disperse more effectively than C. obscurus, and vice versa. Values of da and Vs for E. neoaphidis produced in vitro were substantially larger than those produced in vivo. Thus, in vitro produced conidia would tend to be less effectively dispersed than conidia produced in vivo, suggesting that using mycelia as a source of conidia may be more useful in a mycoinsecticide approach than using infected cadavers. The measurement of spatial and temporal dispersal patterns of entomopathogenic and plant pathogenic fungi in agricultural environments is difficult and time consuming and the results may not be easily applied to situations other than those where they were measured. Mathematical modeling of fungal spore dispersal, based on physical descriptions of atmospheric diffusion processes, offers an alternative method of quantifying
237 dispersal patterns and thus assessing the importance of dispersal in both understanding the development of epizootics and in the management of entomophthoralean fungi as biocontrol agents. Several approaches, based on atmospheric dispersal principles, have been adopted to model the dispersal of plant pathogens within and above crops (McCartney & Fitt 1985) and these could equally be applied to the dispersal of entomopathogens. As all such models require information on the aerodynamic properties of the spores, the results presented here will allow plant pathogen dispersal models to be applied to the dispersal of E. neoaphidis. Such models could be applied to the dispersal and development of epizootics resulting from natural populations or to epizootics produced from the spread of conidia released from in vitro produced material artificially placed within the crop as a biocontrol agent. Thus, the information on the differences in aerodynamic behaviour of in vivo and in vitro produced conidia reported here will allow an assessment of the potential of laboratory produced inoculum of E. neoaphidis to produce epizootics in aphid populations in the field. A C K N O W L E D G E M E N TS F. H. was supported by a PhD research grant from AREEO (Ministry of Agriculture for Iran) and ICARDA (Aleppo, Syria). J. K. P. and H. A. M. are funded by the Ministry of Agriculture, Fisheries and Food, UK. IACR-Rothamsted receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council.
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Aerodynamics of entomophthoralean conidia Modelling of Crop Disease (C. A. Gilligan, ed.) : 107–143. Academic Press London. McCartney, H. A., Schmechel, D. & Lacey, M. E. (1993) Aerodynamic diameter of conidia of Alternaria species. Plant Pathology 42 : 280–286. May, K. R. (1975) An ‘ ultimate ’ cascade impactor for aerosol assessment. Journal of Aerosol Science 6 : 413–419. Morgan, L. W. (1994) Survival, germination responses and infectivity of conidia of Erynia neoaphidis (Zygomycetes : Entomophthorales). PhD thesis, University of Wales, Cardiff. Morgan, L. W., Boddy, L., Clark, S. J. & Wilding, N. (1995) Influence of temperature on germination of primary and secondary conidia of Erynia neoaphidis (Zygomycetes : Entomophthorales). Journal of Invertebrate Pathology 65 : 132–138. Pell, J. K., Eilenberg, J., Hajek, A. E. & Steinkraus, D. S. (2001) Biology, ecology and pest management potential of Entomophthorales. In Fungi as Biocontrol Agents (T. M. Butt, C. Jackson & N. Magan, eds) : 71–153. CAB International, Wallingford.
238 Remaudie' re, G. & Hennebert, G. L. (1980) Re! vision syste! matique de Entomophthora aphidis (Hoffm. in Fres.) Description de deux nouveux pathogens d’aphides. Mycotaxon 11 : 269–321. Sawyer, A. J., Griggs, M. H. & Wayne, R. (1994) Dimensions, density and settling velocity of Entomophthoralean conidia : implications for aerial dissemination of spores. Journal of Invertebrate Pathology 63 : 43–55. Wilding, N. (1973) The survival of Entomophthora spp. in mummified aphids at different temperatures and humidities. Journal of Invertebrate Pathology 21 : 309–311. Wilding, N. & Brady, B. L. (1984) Descriptions of Pathogenic Fungi and Bacteria. Set 82, Nos 812, 814, 815, 817, and 820. Commonwealth Mycological Institute, Kew. Wilding, N. & Brobyn, P. J. (1980) Effects of fungicides on the development of Entomophthora aphidis. Transactions of the British Mycological Society 75 : 279–302. Corresponding Editor : B. Schulz
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DOI : 10.1017\S0953756201005275 Printed in the United Kingdom.
Aerodynamic diameter of conidia of Erynia neoaphidis and other entomophthoralean fungi
Farhad HEMMATI1,3†, Judith K. PELL1*, H. Alastair McCARTNEY2 and Michael L. DEADMAN3‡ " Plant and Invertebrate Ecology Division, # Plant Pathogen Interactions Division, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK. $ Department of Agriculture, University of Reading, Reading RG6 6AT, UK. E-mail : judith.pell!bbsrc.ac.uk Received 5 March 2001 ; accepted 16 September 2001.
The aerodynamic diameter, da, of conidia produced in vivo and in vitro by the entomopathogenic fungus Erynia neoaphidis were estimated using an impaction method. The estimated values of da for conidia produced in vivo were smaller than those produced in vitro : in vivo the values of da for primary and secondary conidia were between 16 and 18 µm (equivalent fall speed, Vs, 0n8–1n0 cm s−") ; for in vitro produced conidia da values were between 28 and 31 µm (Vs, 2n4–3 cm s−"). For conidia produced by field collected cadavers the value of da, was estimated to be similar to that for conidia produced in vivo in the laboratory. The aerodynamic diameter of primary conidia of Conidiobolus obscurus (strain X39) and Zoophthora radicans (strain NW250) produced in vivo were also measured using the same method. The values of da for these two species were 45 and 17 µm (Vs, 6n2 and 0n9 cm s−") respectively. Implications for dispersal of E. neoaphidis are discussed. The physical diameters of the test spores were measured microscopically and compared with the aerodynamic diameters.
INTRODUCTION Entomopathogenic fungi, such as Erynia neoaphidis, are potentially useful biocontrol agents against aphids. However, their effectiveness relies on the early establishment of epizootics within the target insect population. The development of epizootics depends on the ability of the pathogen to spread in time and space. E. neoaphidis infects and kills a number of species of aphid, and spreads by discharging conidia from dead aphids into the air (Hemmati et al. 2001). The conidia are dispersed by wind to other aphid populations where they can initiate infection. The efficiency of dispersal of fungal spores (including those of entomopathogenic fungi), especially within crops, depends on the airflow and on the aerodynamic properties of the spores. The aerodynamic properties of spores determine the rate at which they fall from the air (fall speed) and the rate they are deposited onto surfaces by settling and by impaction (McCartney & Fitt 1985). In most models describing spore dispersal the aerodynamic characteristics of the spores are defined using one parameter * Corresponding author. † Current address : AREEO, Scientific & Research Bureau, PO Box 19835-111, Tehran, Iran. ‡ Current address : Department of Crop Sciences, Sultan Qaboos University, PO Box 34, Al Khod 123, Sultanate of Oman.
called the aerodynamic diameter, da (Legg & Powell 1975, Aylor & Taylor 1983) which is defined as the diameter of a unit density (1 g cm−$) sphere that has the same fall speed, Vs as the spore (May 1975, Chamberlain 1975). The fall speed is also referred to as the settling velocity or terminal velocity and is the speed at which the spore falls through still air when drag and gravity forces balance. The parameter da is used to calculate the rate of deposition of spores onto surfaces by impaction. The fall speed, and hence aerodynamic diameter can be calculated for particles of simple shape, such as spheres or cylinders, if the density of the particle is known (Chamberlain 1975). However, fungal spores rarely have such simple shapes, and even for nearly spherical spores da is often less than the diameter, d (Gregory 1973, McCartney 1991). E. neoaphidis actively discharges conidia (primary conidia) from aphid cadavers. After deposition primary conidia can actively discharge smaller, secondary conidia. Primary conidia are lemon-shaped, and secondary conidia are either of similar shape or more rounded (Morgan et al. 1995). The size range of primary conidia has been reported to be between 18–35 µm long and 11–15 µm wide and the range of the length\diam ratio (L\D) as 1n7–2n3. For secondary conidia, the equivalent ranges are : length 14–24 µm, width 11–20 µm and L\D 1n1–1n4 (Remaudie' re & Hennebert 1980, Wilding & Brady
Aerodynamics of entomophthoralean conidia 1984, Brown 1985). Morgan (1994) reported that the size of primary conidia (isolate X4) produced in vitro were significantly larger (mean 32i15n4 µm) than those produced in vivo (mean 19n8i11n8 µm). Eilenberg, Bresciani & Latge! (1990) showed that primary conidia of Entomophthora schizophorae produced in vitro were larger than those produced in vivo. As far as we are aware, there are no reports of values of Vs for E. neoaphidis conidia. Furthermore Vs has been measured for spores of only a few entomopathogenic fungi. For example, Sawyer, Griggs & Wayne (1994) measured Vs of Zoophthora radicans (isolates ARSEF 2282 and 2283), Conidiobolus thromboides (ARSEF 40) and C. obscurus (ARSEF 70) and found mean values of 0n36, 0n37, 1n39 and 4n49 cm s−", respectively. The results of measurements of the aerodynamic diameter of primary and secondary conidia of E. neoaphidis, which were produced both in vivo and in vitro, are reported. Measurements of da and Vs of in vivo-produced conidia of Conidiobolus obscurus and Zoophthora radicans are also presented. MATERIALS AND METHODS Conidia sources Sources of in vivo conidia Two sources of Erynia neoaphidis conidia were used. One was cadavers of pea aphids, Acyrthosiphon pisum, infected in the laboratory using the methods of Wilding (1973) with an isolate of E. neoaphidis (S1 ; from the Rothamsted culture collection). This isolate was originally collected from the nettle aphid, Microlophium carnosum on the Rothamsted farm. The other source was E. neoaphidis-infected, dried cadavers of rose-grain aphids, Metopolophium dirhodum, collected from naturally occurring epizootics in wheat fields on Rothamsted farm. Acyrthosiphon pisum aphids were infected with Conidiobolus obscurus (X39 from the Rothamsted culture collection) and diamondback moths, Plutella xylostella, were infected with Zoophthora radicans (reference NW 250 R (Malaysia) from the Rothamsted culture collection) in the laboratory. Infected cadavers of each insect species were used as sources of conidia for these species of fungi. Conidia sources for each experiment consisted of ten sporulating cadavers of the test species. The infected cadavers were placed on the base of a 50 ml, 2n5 cm diam, centrifuge tube lined with wet tissue paper. The tube was incubated at 10 mC and 100 % RH in darkness overnight to encourage sporulation. Source of in vitro conidia Erynia neoaphidis (S1) was grown on Sabouraud dextrose agar supplemented with milk and egg yolk (Wilding & Brobyn 1980) at 20 m in darkness for 3–4 wk prior to experimentation. Sections of mycelia cut from the growing edge of the colony were placed inside the
234 centrifuge tubes and incubated at 10 m, 100 % RH in darkness for 12 h, to encourage sporulation. Secondary conidia were produced from primary conidia from both in vitro and in vivo sources in the same way. Primary conidia were collected for 6 h in Petri dishes containing 1 % water agar, at 20 m and 100 % RH. A section of water-agar containing primary conidia was then placed in a 50 ml centrifuge tube. The primary conidia sporulated on the water agar providing a source of secondary conidia. The impaction method for estimating aerodynamic diameter (see below) was also tested using Lycopodium clavatum and Calvatia gigantea (giant puff-ball) spores. Lycopodium clavatum spores were obtained, as dry spores, from BDH Laboratory Chemicals (Poole, UK) and C. gigantea spores were obtained from a mature giant puff-ball collected on Rothamsted farm. Estimation of the aerodynamic diameter and settling velocity of conidia The aerodynamic diameter of test conidia was estimated from their impaction characteristics using the method described by McCartney, Schmechel & Lacey (1993). This method uses a May Ultimate Impactor (MUI, Research Engineers, London ; May 1975). The MUI is a cascade impactor used to classify particles according to their aerodynamic characteristics. Air is drawn through a series of seven stages, each consisting of a jet formed by a slit held above a removable microscope slide. Each stage collects smaller particles than its predecessor. For a uniformly sized sample, the proportion, P, of the total sample deposited on each stage depends only on the aerodynamic diameter of the particles (McCartney et al. 1993). For the first stage, P increases monotonically with aerodynamic size of the particles, but for the other stages, P increases to a maximum value then declines. The mean aerodynamic diameter of a sample can be estimated if the values of P for each stage are measured (McCartney et al. 1993). For each experiment, conidia from the test source were introduced into the MUI by inverting the centrifuge tube containing the source above the inlet : sporulating cadavers (primary conidia), sporulating mycelia (primary conidia), or sporulating primary conidia (secondary conidia). Nylon netting placed across the mouth of the centrifuge tube ensured that the source remained inside the tube and only the conidia passed into the MUI. The MUI was operated for 3 h with sources producing primary conidia and 15 h for sources producing secondary conidia. Microscope slides used in the MUI were coated with a layer of a mixture of petroleum jelly (Vaseline) and paraffin wax (100 : 18 w\w ; British Aerobiology Federation 1996) to retain conidia. After exposure, slides were removed from the MUI and the numbers of conidia collected on five identical traverses across the whole width of each slide were counted. One traverse was across the centre of the trace, the others were 1 and
F. Hemmati and others
235
2 cm on either side of the central traverse. The traverse width was 1 mm, the magnification i100 and the distance between the traverses 5 mm. The counts were used to estimate the total number of conidia deposited in the MUI and the proportion, P, collected on each stage. The average value of the aerodynamic diameter, da for sampled conidia in each test was estimated from the measured values of P for each stage using the methods described in McCartney et al. (1993). Briefly, the mean value of da was taken as the value that minimised the chi-squared value of P (i.e. (PmeasuredkPexpected)#\ Pexpected) when summed over all the stages which collected conidia. Pmeasured were the values of P measured for each stage, and Pexpected were the theoretical values for a monodisperse sample of aerodynamic diameter da (McCartney et al. 1993). Settling velocity (Vs) was determined from Chamberlain (1975) : (1) Vs l 0n00308 d#a where da is in µm and Vs is in cm s−". All experiments were done at 20 mC and two trials (replicates) were made for each conidium type, except for Erynia neoaphidis when two additional trials were done with laboratory produced cadavers (in vivo). Further trials were done using Lycopodium clavatum and Calvatia gigantea spores. The aerodynamic properties of these spores are known and the tests were done as an additional check on the method. The maximum width and length of conidia of each entomopathogen was estimated by measuring 50 conidia from each treatment using a micrometer graticule at i500 magnification. Condia were selected randomly by moving the stage across the slide and selecting conidia from the centre of the field from those caught on all stages of the MUI. RESULTS There was some variation in the size and shape of
conidia for the three entomopathogenic species tested (Table 1). Both primary and secondary conidia derived from E. neoaphidis (S1) cultured in vitro were longer and wider, but had a shorter length\width ratio, than in vivo conidia derived from pea aphid cadavers (Table 1). The mean values of length (L ), width (W ) and L\W were statistically different (t-test) for the three species. The mean length and width of primary conidia produced from field collected cadavers was significantly larger (P 0n05) than that of those produced in vivo in the laboratory, but significantly shorter (P 0n05) than those produced in vitro. However, E. neoaphidis conidia produced by Metopolophium dirhodum cadavers collected in the field, tended to be more elongated than the others (mean L\W values were 1n98, 1n89 and 1n67 for field in vivo, laboratory in vivo and laboratory in vitro respectively, and all were significantly different to each other, P 0n05). Secondary conidia tended to be smaller than primary conidia, except that the widths of primary and secondary conidia produced by the S1 strain in vivo were not significantly different (P l 0n5). Primary conidia of Zoophthora radicans produced from cadavers of Plutella xylostella tended to be shorter and narrower, but more elongated than E. neoaphidis conidia, while primary conidia of Conidiobolus obscurus, produced from Acyrthosiphon pisum, were larger, but more spherical (Table 1). In all experiments, most conidia were collected on stages 1–4 of the impactor (Table 2). Conidia of C. obscurus were found mostly on stages 1 and 2. In all experiments, the estimated aerodynamic diameter of conidia was greater than their mean maximum width and length, except for the primary conidia of fieldcollected E. neoaphidis in which the aerodynamic diameter was larger than the conidia’s width, but was smaller than their length (Table 3). Primary conidia of C. obscurus had the largest estimated value of da and in vivo produced conidia of E. neoaphidis had the smallest. Both primary and secondary conidia of E. neoaphidis (S1) cultured in vitro had larger estimated aerodynamic diameters than those cultured in vivo (Table 3).
Table 1. Dimensions of conidia of test fungi. Conidia-source Erynia neoaphidis (S1), in vivo, primary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), in vivo, primary Zoophthora radicans (NW250), in vivo, primary
Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range Mean () Range
Length (µm)
Width (µm)
Length\width ratio
18n8 (2n9) 13–25n9 16n5 (2n3) 11n1–22n2 26n7 (2n3) 22n2–31n5 21n6 (2n8) 14n8–27n8 23n5 (2n2) 18n5–29n6 33n4 (2n3) 28n9–40n0 15n9 (2n2) 11n1–20n6
10n2 (1n6) 7n4–16n7 10n4 (1n6) 7n4–14n8 16n2 (2n1) 13n0–20n6 17n1 (1n7) 13n0–20n6 12n0 (1n3) 9n3–14n8 27n8 (2n0) 23n3–32n2 7n3 (1n1) 5n6–11n1
1n89 (0n39) 1n11–2n78 1n61 (0n23) 1n19–2n01 1n67 (0n21) 1n30–2n17 1n27 (0n16) 1n0–1n89 1n98 (0n27) 1n50–2n50 1n21 (0n06) 1n06–1n33 2n20 (0n38) 1n49–3n68
Aerodynamics of entomophthoralean conidia
236
Table 2. Percentage of conidia collected in each stage of the May Ultimate Impactor. Stage no.
Species (isolate in parenthesis)
Trial
Total number of conidia counted
1
2
3
4
5
6
7
Erynia neoaphidis (S1), in vivo, primary E. neoaphidis" (S1), in vivo, primary & secondary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), in vivo, primary Zoophthora radicans (NW250), in vivo, primary
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
42 971 29 360 6884 1129 6106 5455 15 562 13 265 19 668 4294 9273 11 610 1461 2576 8014 14 974
8n56 10n69 2n96 5n31 3n21 8n05 24n70 32n59 25n41 30n79 10n50 10n79 94n46 92n97 2n35 2n86
31n10 27n02 42n18 71n80 70n29 59n82 35n36 34n38 32n74 47n74 49n29 40n72 5n41 5n36 44n75 41n83
39n43 43n41 36n7 17n98 23n58 30n63 32n52 28n90 34n40 21n15 34n32 42n33 0n14 1n67 40n40 41n12
16n40 13n75 13n49 4n87 2n01 1n17 3n53 3n05 6n34 0n30 3n56 2n43 0 0 11n21 13n26
3n59 4n07 4n31 0 0n72 0n16 0n76 0n96 0n94 0n02 1n23 1n90 0 0 0n80 0n77
0n84 0n77 0n33 0 0n18 0n16 0n13 0n13 0n18 0 1n05 1n68 0 0 0n37 0n13
0n07 0n28 0 0 0 0 0 0 0 0 0n05 0n14 0 0 0n12 0n03
" The same source was used for each trial and the trials were done consecutively. No discrimination was made between primary and secondary conidia ; all conidia were counted.
Table 3. Aerodynamic characteristics of conidia estimated from the deposit on the May Ultimate Impactor. Aerodynamic diameter, da and fall speed, Vs. Trial 1
Trial 2 χ#
Conidia source
Mean LiW (µm)
da (µm)
da (µm)
Erynia neoaphidis (S1), in vivo, primary E. neoaphidis" (S1), in vivo, primary & secondary E. neoaphidis (S1), in vivo, secondary E. neoaphidis (S1), in vitro, primary E. neoaphidis (S1), in vitro, secondary E. neoaphidis (Field), in vivo, primary Conidiobolus obscurus (X39), primary Zoophthora radicans (NW250), primary
18n8i10n2 —
16 17
6n5 8n7
16 18
16n5i10n4 26n7i16n2 21n6i17n1 23n5i12 33n4i27n8 15n9i7n3
18 29 30 17 45 17
9n6 15n9 17n5 0n4 0n01 9n3
17 31 28 17 45 17
Trial 1 Vs (cm s−")
Trial 2 Vs (cm s−")
4n2 5n8
0n8 0n9
0n8 1n0
3n3 10n9 4n6 0n5 0n03 9n7
1n0 2n6 2n8 0n9 6n2 0n9
0n9 3n0 2n4 0n9 6n2 0n9
χ#
" Experiments were made consecutively using the same source ; all conidia were counted.
DISCUSSION Experiments were done with two in vivo sources of Erynia neoaphidis, one cultured in the laboratory on pea aphids (Acyrthosiphon pisum) and one derived from field collected cadavers of rose grain aphids (Metopolophium dirhodum). Although the conidia derived from field collected cadavers were larger and more elongated than those produced from pea aphids, the estimated values of da were similar. This suggests that dispersal characteristics for these conidia would also be similar. The aerodynamic diameters of E. neoaphidis conidia were closer in size to the length of the conidia than to their width. For non-spherical particles, aerodynamic diameters are expected to be smaller than the largest dimension, but larger than the smaller dimension. For example, for elongated conidia of Alternaria species, da was found to range between about 10 and 40 µm while the length varied between about 40 and 150 µm
(McCartney et al. 1993). Aerodynamic diameters are defined as the diameter of a unit density (1 g cm−$) sphere with the same aerodynamic characteristics. Thus the values of da estimated for E. neoaphidis conidia would suggest that their densities were greater than that of water. Alternatively, as da values were estimated using impaction, the uneven distribution of mass within the conidia combined with its shape may have enhanced the impaction efficiency of the conidia by orientating the conidia to reduce drag, this would have also increased the estimate of da. The results of additional tests using Lycopodium clavatum and C. gigantea spores to check the impaction method, suggested that the impaction method did not overestimate da values. Estimates of da for L. clavatum spores were between 25 and 27 µm, slightly less than the measured diameter of about 30 µm, and about 5 µm for 4n5 µm diameter C. gigantea spores. It therefore seems unlikely that the impaction method significantly overestimated the aerodynamic diameter of the E. neoaphidis conidia.
F. Hemmati and others The aerodynamic diameters of Conidiobolus obscurus and Zoophthora radicans estimated in these experiments were larger than their physical dimensions. This also suggests that the densities of these spores were greater than that of water or that their shape or mass distribution enhanced their impaction efficiencies. Conidia of both species are not spherical. The fall speeds corresponding to estimated da were larger than those measured by Sawyer et al. (1994). They measured fall speed by direct observation of conidia falling in narrow glass tubes and noted that the conidia of C. obscurus, in particular, fell more slowly than predicted by Stokes’ law. Their results may have been influenced by either wall (Edmonds 1979) or electrostatic effects. With the exception of species of Massospora, all entomophthoralean fungi produce actively discharged conidia, which is clearly an adaptation for dispersal by wind (Hemmati et al. 2001, Pell et al. 2001). An understanding of the wind-borne dispersal of such fungi may provide an insight into how they have evolved to exploit different ecological niches within the agro-environment. For example, for the two aphid pathogens E. neoaphidis and C. obscurus, da for E. neoaphidis is smaller than that for C. obscurus and so it is better adapted to aerial dispersal. C. obscurus produces resting spores for survival in the absence of the host and these are not known in E. neoaphidis. This suggests that even if E. neoaphidis causes local extinctions in its host population it is able to disperse to other host populations. An understanding of spore dispersal capabilities of entomopathogenic fungi is also useful in assessing their suitability in different biological control strategies. For example, if the target pest is patchily distributed or alternate hosts are being exploited as reservoirs of a specific fungus then it is essential that the fungus can disperse between those populations, particularly between reservoir and target populations. However, if a fungus is to be applied as a mycoinsecticide to an area of crop, with the goal of initiating a large epizootic rapidly in the local area, then a species which is deposited rapidly would be more effective. Thus, E. neoaphidis may be more suited to the first type of strategy than C. obscurus : because of its smaller da it would disperse more effectively than C. obscurus, and vice versa. Values of da and Vs for E. neoaphidis produced in vitro were substantially larger than those produced in vivo. Thus, in vitro produced conidia would tend to be less effectively dispersed than conidia produced in vivo, suggesting that using mycelia as a source of conidia may be more useful in a mycoinsecticide approach than using infected cadavers. The measurement of spatial and temporal dispersal patterns of entomopathogenic and plant pathogenic fungi in agricultural environments is difficult and time consuming and the results may not be easily applied to situations other than those where they were measured. Mathematical modeling of fungal spore dispersal, based on physical descriptions of atmospheric diffusion processes, offers an alternative method of quantifying
237 dispersal patterns and thus assessing the importance of dispersal in both understanding the development of epizootics and in the management of entomophthoralean fungi as biocontrol agents. Several approaches, based on atmospheric dispersal principles, have been adopted to model the dispersal of plant pathogens within and above crops (McCartney & Fitt 1985) and these could equally be applied to the dispersal of entomopathogens. As all such models require information on the aerodynamic properties of the spores, the results presented here will allow plant pathogen dispersal models to be applied to the dispersal of E. neoaphidis. Such models could be applied to the dispersal and development of epizootics resulting from natural populations or to epizootics produced from the spread of conidia released from in vitro produced material artificially placed within the crop as a biocontrol agent. Thus, the information on the differences in aerodynamic behaviour of in vivo and in vitro produced conidia reported here will allow an assessment of the potential of laboratory produced inoculum of E. neoaphidis to produce epizootics in aphid populations in the field. A C K N O W L E D G E M E N TS F. H. was supported by a PhD research grant from AREEO (Ministry of Agriculture for Iran) and ICARDA (Aleppo, Syria). J. K. P. and H. A. M. are funded by the Ministry of Agriculture, Fisheries and Food, UK. IACR-Rothamsted receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council.
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