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Influence of Substrate Type and Temperature on the Developmental Morphology ofPandora neoaphidis(Zygomycetes: Entomophthorales), a Pathogen of the Tobacco Aphid (Homoptera: Aphididae)
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
72, 112–118 (1998)
IN984765
Influence of Substrate Type and Temperature on the Developmental Morphology of Pandora neoaphidis (Zygomycetes: Entomophthorales), a Pathogen of the Tobacco Aphid (Homoptera: Aphididae) Surendra K. Dara1 and Paul J. Semtner Southern Piedmont Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University, Route 3 Box 60, Blackstone, Virginia 23824 Received November 4, 1996; accepted February 5, 1998
Developmental morphology of Pandora neoaphidis was observed on the surfaces of the tobacco aphid, Myzus nicotianae, tobacco leaves (Nicotiana tabacum), and glass coverslips at 13 and 20°C for 12 and 24 h postinoculation. Pandora neoaphidis responded similarly on the two living substrates, but differed on the inert coverslips. The proportions of ellipsoid conidia (primary and secondary) were similar on all substrates. Higher proportions of appressoria and lower proportions of round secondary conidia and germinating conidia occurred on the aphids and leaves than on the coverslips. Appressoria predominated over round secondary conidia and germinating conidia on the living substrates at 20°C, but the opposite was seen at 13°C. The proportions of ellipsoid conidia were similar at both temperatures. On coverslips, the proportions of appressoria and round secondary conidia were similar at both temperatures. However, the proportions of germinating and ellipsoid conidia were higher at 13 and 20°C, respectively. r 1998 Academic Press Key Words: Pandora neoaphidis; Myzus nicotianae; substrate; temperature; germination; tobacco leaf.
The fate of conidia after they land on the host surface determines the success of invasion by the pathogen. Brobyn and Wilding (1977) and Butt et al. (1990) studied the invasive and developmental processes of P. neoaphidis on Acyrthosiphon pisum. The primary conidium of P. neoaphidis germinates about 4 h postinoculation and forms a germ tube, an infective appressorium, or a secondary conidium (Brobyn and Wilding, 1977; Butt et al., 1990). Differential responses have been demonstrated for P. neoaphidis in conidial germination on coverslips and leaves of field beans (Brobyn et al., 1987). Fransen (1995) demonstrated the response of Aschersonia aleyrodis, another entomopathogen, on cucumber leaves and the cuticle of Trialeurodes vaporariorum (Fransen, 1995). Information on the response of P. neoaphidis to the type of substrate will provide a greater understanding of the fate of inoculum on different surfaces in the environment. This research examined the developmental morphology of conidia of P. neoaphidis inoculated on three surfaces and incubated at two temperatures. MATERIALS AND METHODS
INTRODUCTION
Rearing Aphids
The host range of Pandora neoaphidis includes several species of aphids and a few species of hemipterans (Wilding and Brady, 1984). Its germination, type of conidia, growth in vitro, and production of conidia in vivo are influenced by temperature (Morgan et al., 1995; Yu et al., 1995). Optimal temperatures for conidial germination of P. neoaphidis are between 18 and 23°C (Morgan et al., 1992, 1995), while maximum conidial production occurs between 10 and 25°C (Yu et al., 1995). 1Current address: International Institute of Tropical Agriculture, 08 BP 0932 Tri Postal, Cotonou, Republic of Benin, West Africa. E-mail: [email protected].
0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Tobacco aphids, Myzus nicotianae, were collected from field tobacco, Nicotiana tabacum, and reared on tobacco plants growing in sterile potting medium in a growth chamber at 22°C and 16L:8D photophase. Newly born aphids were collected from these plants with a camel’s hair brush and transferred to excised leaves from tobacco grown in the greenhouse. The leaves were rinsed with deionized water and air dryed. These leaves were placed in Styrofoam cups (710 ml) and their petioles were inserted in 1% water agar (Fisher Scientific, Springfield, NJ) in the bottom of the cups. Lids were placed on the cups to keep the aphids inside. Aphids were transferred to new leaves after 5 or 6 days. When they were 9 days old, aphids were again trans-
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ferred to new leaves in new cups and allowed to deposit nymphs for 6–8 h. Viviparae were then removed using a moist camel’s hair brush and neonate nymphs were retained in the cups. Nymphs from these cultures were used in the experiment when they were 5 days old. Culturing P. neoaphidis To culture P. neoaphidis, infected tobacco aphids were collected from the field, surface sterilized for 30 to 60 s each in a 5–10% bleach (sodium hypoclorite 5.25%) and a 0.01% gentamicin solution, and transferred onto modified SEMA medium (G. L. Nordin personal communication; Yu et al., 1995). Ingredients of SEMA included 74.6% fresh yolk from unwashed eggs, 14.4% vitamin D milk, 11% Sabouraud maltose agar (Difco Laboratories, Detroit, MI), 0.1% gentamicin (Sigma Chemical Co., St. Louis, MO), and 0.05% penicillin (Agri Laboratories Ltd., St. Joseph, MO). These cultures were incubated at 20°C and 14L:10D photophase. The pathogen used for the experiment had been subcultured four times in vitro after it was isolated from the aphid host. Two- to 3-week-old cultures from a common source were used for the experiment. Sporulating cultures were selected for the experiment based on the conidial deposition on the lids of Petri plates. Inoculating Aphids, Tobacco Leaves, and Coverslips On the day of the experiment, 25 aphids, 5 days old, were placed on a separate tobacco leaf disc (about 55 mm dia) in each of four small Petri plates (60 3 15 mm). Before the discs were cut, tobacco leaves from greenhouse plants were rinsed with deionized water. A filter paper (Whatman No. 1, 55-mm circle) was placed on the bottom and a similar one was attached to the lid of each Petri plate with nontoxic Permanent Glue Stic (Avery Dennison, Framingham, MA). These filter papers were moistened to maintain the high humidity ($98%) required for conidial germination (Wilding, 1971; Carruthers and Hural, 1990; Yu et al., 1995). In each Petri plate, five small pieces of coverslip (about 40 mm2 ) were placed on the leaf disc, one in the center and one in each of four outer quadrants. Large Petri plates (100 3 15 mm) containing fungal cultures were inverted over the small plates to expose aphids, leaf discs, and coverslips to conidial showers from the cultures. The plates were then covered with moist paper towels, placed in a sealed plastic bag, and inoculated in the dark at 20°C. Relative humidity inside the plastic bags was measured with a hygrometer (127 mm diam by 32 mm) (Abbeon Cal. Inc., Santa Barbara, CA). Moist paper towels helped to maintain the relative humidity at $98% in the plastic bags. Aphids were exposed to conidial showers for 4 h. During inoculation, culture plates were rotated 90° every 60 min to promote uniform conidial showers over
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the exposed area. After inoculation, cultures were removed and lids were placed on the small plates. Two plates each were incubated in the dark at 13 and 20°C. At 12 and 24 h postinoculation, one plate each was taken from the 13 and 20°C chambers, respectively. The aphids from each Petri plate were divided into five groups of five aphids. Five small pieces (about 40 mm2 ) were cut from the tobacco leaf disc in each Petri plate to represent five samples of each treatment. Thus each temperature and time treatment (Petri plate) contained five groups of aphids and five pieces each of tobacco leaf and coverslip. A group of five aphids, a piece of tobacco leaf, and a piece of coverslip represented a replication from a section of a leaf disk. SEM The surfaces of aphids, pieces of tobacco leaf, and coverslips were critical point dried and sputter-coated with gold–palladium (10 nm) for scanning electron microscopy (SEM). The conidial germination was observed under SEM (Philips 505) on the surfaces of tobacco aphid, tobacco leaf, and coverslip. Observations were made on the entire dorsal region of each tobacco aphid (ca. 1 to 2 mm2 ) and at five random locations (area of each location about 0.2 mm2 ) on each piece of tobacco leaf and coverslip. Statistical Analysis Analysis of variance (GLM procedure, SAS Institute, 1994) was used to analyze all data. Significantly different means for substrate were separated by Tukey’s studentized range test (SAS Institute, 1994). Surface effects were analyzed separately within each temperature 3 time treatment. In addition, comparisons were made for treatments over time and over temperature. The proportions of different categories of structures found on three substrates were calculated. Data were arcsine-transformed to stabilize variances. RESULTS
Conidial germination as indicated by germ tubes, appressoria, or secondary conidia occurred on all substrates. Structures found on the three substrates were placed in the following categories: nongerminated ellipsoid conidium (Fig. 1), germinated conidium with collapsed spore and intact germ tube (Fig. 2), conidium that produced an appressorium (Fig. 3), conidium with penetration peg (Fig. 4), round secondary conidium (Fig. 5), secondary conidium developing on a germ tube (Fig. 6), ellipsoid conidium germinating by means of germ tube or initiation of successive (secondary or tertiary) conidium or both (Fig. 7), and round secondary conidium germinating by means of germ tube (Fig. 8). Primary conidia and secondary conidia that resembled
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primary conidia were considered as ellipsoid conidia since the two types of conidia were often difficult to separate. Germination of conidia was influenced by substrate, temperature, and time (Table 1). The relative proportion of different structures formed during germination varied on the three substrates and at the two temperatures when observed at two time intervals (P , 0.05).
interaction: df 2, F 5 6.73, P 5 0.0031). Time had no effect at either temperature on the proportion of appressoria on aphids and at 20°C on coverslips and leaves. However, at 13°C significantly higher (P 5 0.01) proportions of appressoria were found at 24 h than at 12 h postinoculation on coverslips and leaves (temperature 3 time interaction: df1, F 5 6.35, P 5 0.0160). Ellipsoid Conidium
Germ Tube The proportions of germ tubes observed on the three substrates were not significantly different (results not shown). Temperature did not influence germ tubes on aphids and coverslips, but significantly higher (P 5 0.0001) proportions of germ tubes were found on leaves at 20 than at 13°C. Time did not influence the formation of germ tubes on aphids, but significantly higher (P , 0.05) proportions of germ tubes were seen at 24 h than at 12 h postinoculation for both temperatures on leaves and coverslips.
The overall proportion of nongerminated ellipsoid conidia was similar on all substrates (Table 1). However, the proportion was significantly higher on coverslips (P 5 0.0002) at 20 than at 13°C. Time had no influence on ellipsoid conidia at both temperatures on aphids, at 13°C on coverslips, and at 20°C on leaves. The proportion of ellipsoid conidia significantly decreased (P 5 0.01) 24 h postinoculation at 20°C on coverslips and at 13°C on leaves (surface 3 temperature 3 time interaction: df 2, F 5 4.34, P 5 0.0199). Round Secondary Conidium
Penetration Peg Penetration was seen at 20°C on the abdomen of an aphid within 12 h postinoculation. Since there was only one penetration peg in the entire study, it was excluded from the analysis. Germinating Round Secondary Conidium Germinating round secondary conidia were found only 24 h postinoculation at both temperatures on coverslips and at 20°C on leaves. They were not seen on aphids. Secondary Conidium on Germ Tube Germ tubes bearing secondary conidia were not seen on aphids. They occurred on coverslips at both temperatures and on leaves at 20°C. However, germ tubes were not observed within 12 h postinoculation on coverslips at 13°C and on leaves at 20°C. Since no sign of successive development was seen in germ tubes with collapsed conidia, the remaining structures were regrouped into four categories viz., appressoria, ellipsoid conidia, round secondary conidia, and germinating conidia (including germinating ellipsoid and round secondary conidia and conidia on germ tubes) and compared (Table 1). Appressorium Pandora neoaphidis produced similar proportions of appressoria on aphids and leaves, but proportions were significantly lower (P 5 0.0001) on the inert coverslips (Table 1). Formation of appressoria was significantly higher (P , 0.05) at 20 than at 13°C on aphids and leaves, but not on coverslips (surface 3 temperature
The majority of round secondary conidia on all substrates were still attached to the collapsed primary conidia where they were produced. The production of round secondary conidia was significantly higher (P 5 0.002) on coverslips than on aphids or leaves (Table 1). Proportions of round secondary conidia were influenced by temperature on aphids and leaves, but not on coverslips. On leaves and aphids, significantly lower (P 5 0.0001) proportions of round secondary conidia were seen at 20 than at 13°C (surface 3 temperature interaction: df 2, F 5 6.64, P 5 0.0033). A significantly higher proportion of round secondary conidia was seen at 24 h than at 12 h postinoculation at 20°C on coverslips, and at 13°C on leaves (surface temperature 3 time interaction: df 2, F 5 6.6, P 5 0.0034). Germinating Conidium Significantly higher proportions of germinating ellipsoid conidia were seen on coverslips than on aphids and leaves, and at 13 than at 20°C on all substrates (surface 3 temperature interaction: df 2, F 5 3.5, P 5 0.04). The proportion of germinating ellipsoid conidia was higher at 12 h than at 24 h postinoculation on all substrates at 13°C; however, the differences were not significant on aphids (temperature 3 time interaction: df1, F 5 20.68, P 5 0.0001). Similar proportions of germinating ellipsoid conidia were seen on all substrates at both time intervals at 20°C. DISCUSSION
Stimuli from the substrate induce formation of appressoria in entomopathogenic fungi (St. Leger, 1993). Our observations indicate that cues from the living
SUBSTRATE AND TEMPERATURE EFFECTS ON Pandora neoaphidis
FIGS. 1–4.
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Scanning electron micrographs.
substrates are important in the formation of appressoria in P. neoaphidis. The proportions of appressoria were significantly higher on the living substrates than on the inert coverslips. The production of appressoria in P. neoaphidis precedes penetration of the host cuticle and is in response to the recognition that the substrate is suitable for infection (Butt et al., 1990). Other entomopathogenic fungi form appressoria in response to nutritional and physical stimuli. Various levels of nitrogenous nutrients, glucose, and hard, hydrophobic surfaces induce in vitro formation of appressoria in Metarhizium anisopliae (St. Leger et al., 1989; St. Leger, 1993). Appressorial formation by Zoophthora radicans is severely depressed in vitro in the absence of nitrogen and carbon sources (Magalhaes et al., 1991). When conditions are not favorable for infection, the fungus continues to produce successive stages of conidia, until the nutrients are depleted. In our study,
P. neoaphidis expressed this behavior by producing significantly higher proportions of round secondary conidia and germinating conidia and less of appressoria on the inert coverslips. In contrast, the pathogen produced more infective appressoria and less successive stages of development on aphid and leaf surfaces, while the proportion of ellipsoid conidia remained constant on all substrates. Apparently P. neoaphidis did not receive the signal to produce infective structures on coverslips and continued to germinate by other means. Brobyn et al. (1987) reported that conidia of P. neoaphidis rapidly lost their infectivity at 100% relative humidity on coverslips due to exhaustion of energy reserves from production of successive generations of secondary conidia in a short period. The conidia of A. aleyrodis, another entomopathogenic fungus, had lower germination on the surface of cucumber leaf than on the cuticle of fourth-instar
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FIG. 1. Ellipsoid primary or secondary conidia on aphid cuticle (bar 5 10 µm). FIG. 2. Germinated conidium with collapsed spore (arrowhead) and intact germ tube (arrow) on coverslip (bar 5 100 µm). FIG. 3. Conidium (arrowhead) with globose appressorium (arrow) on aphid cuticle (bar 5 10 µm). FIG. 4. Conidium (arrowhead) with penetration peg (arrow) on the abdomen of aphid (bar 5 10 µm). FIGS. 5–8. Scanning electron micrographs. FIG. 5. Round secondary conidium (arrow) singly and on collapsed primary conidium (arrowhead) on coverslip (bar 5 10 µm). FIG. 6. Secondary conidium (arrow) developing on a germ tube (arrowhead) on coverslip (bar 5 10 µm). FIG. 7. Germinating ellipsoid conidia. (A) Ellipsoid conidia (arrowhead) with different stages of development of secondary/tertiary conidia (arrow) (bar 5 10 µm), (B) ellipsoid conidium with germ tube (bar 5 10 µm), (C) ellipsoid conidium with initiation of secondary/ tertiary conidium and germ tube (bar 5 10 µm) on aphid cuticle. FIG. 8. Germinating round secondary conidium, on coverslip (bar 5 100 µm). Round secondary conidium (arrowhead) with a germ tube (arrow) on a collapsed primary conidium.
larvae of greenhouse whitefly, T. vaporariorum (Fransen, 1995). This reduced germination was attributed to lack of stimulation or presence of inhibitory factors on cucumber leaves. Germination of P. neoaphidis conidia was slower on leaves of field beans (Vicia faba) than on coverslips at 70–77% relative humidity (Brobyn et al., 1987). Brown et al. (1995) reported from in vitro experiments that conidial germination of P. neoaphidis was inhibited by volatiles from tobacco leaves, especially if the tobacco leaf has supported a previous infestation of tobacco aphids. They also re-
ported that aphid infection rates were not affected by green leaf volatiles. These results led them to hypothesize that there is little inhibition when the conidia are in direct contact with the aphid cuticle. In our study, conidial germination was similar on aphid cuticle and tobacco leaf. Unknown factors may have stimulated appressorial formation or simulated aphid cuticle on tobacco leaf surface. Similarities between the convoluted topography of aphid cuticle and tobacco leaf surface may have caused the similar responses of P. neoaphidis on those two surfaces. The
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TABLE 1 Relative Proportion of Appressoria, Ellipsoid Conidia, Round Secondary Conidia, and Germinating Conidia Mean 6 SE Effect
Temp. (°C)
Time
APP a
EC
RSC
GC
Substrate b Aphid Coverslip Leaf Temperature c Aphid Aphid Coverslip Coverslip Leaf Leaf Time d Aphid Aphid Aphid Aphid Coverslip Coverslip Coverslip Coverslip Leaf Leaf Leaf Leaf
13 20 13 20 13 20 13 13 20 20 13 13 20 20 13 13 20 20
12 24 12 24 12 24 12 24 12 24 12 24
22.6 6 4.3a 4.6 6 1.4b 22.0 6 3.8a
54.3 6 3.9a 50.2 6 3.4a 56.6 6 3.0a
9.9 6 1.9b 16.4 6 2.2a 8.7 6 2.3b
13.3 6 3.5b 28.9 6 2.7a 12.8 6 2.1b
14.0 6 4.1** 35.5 6 5.7 4.5 6 1.7NS 4.7 6 2.3 11.5 6 3.9** 32.5 6 4.5
50.9 6 5.1NS 59.3 6 5.9 40.8 6 2.0*** 59.6 6 5.1 53.1 6 4.0NS 60.1 6 4.4
13.2 6 2.3** 5.0 6 1.7 16.5 6 2.6NS 16.2 6 3.7 16.7 6 2.8*** 0.7 6 0.2
22.0 6 3.4*** 0.2 6 0.2 38.3 6 2.2*** 19.5 6 2.5 18.8 6 3.0*** 6.8 6 1.1
7.2 6 2.5NS 22.4 6 6.8 37.1 6 11.8NS 33.9 6 4.5 0.2 6 0.1* 8.7 6 1.8 0.9 6 0.5NS 8.6 6 3.9 0.9 6 0.2** 22.1 6 3.4 33.7 6 8.4NS 31.3 6 4.5
50.7 6 2.5NS 51.1 6 12.0 61.2 6 12.5NS 57.5 6 3.8 39.5 6 1.8NS 42.0 6 3.6 72.9 6 3.9** 46.3 6 3.6 61.8 6 5.0* 44.4 6 3.4 60.7 6 7.6NS 59.4 6 5.5
13.6 6 3.7NS 12.6 6 3.1 1.3 6 0.7NS 8.6 6 0.9 17.8 6 1.6NS 15.2 6 5.2 7.4 6 2.1** 25.0 6 4.4 11.1 6 2.4* 22.3 6 3.7 0.4 6 0.2NS 0.9 6 0.4
28.5 6 1.6NS 13.9 6 4.9 0.4 6 0.4NS 0.0 6 0.0 42.4 6 2.4NS 34.1 6 2.6 18.8 6 3.5NS 20.2 6 3.8 26.3 6 2.9* 11.2 6 2.2 5.2 6 1.2NS 8.3 6 1.7
a APP, appressorium 1 penetration; EC, ellipsoid primary or secondary conidium; RSC, round secondary conidium; GC, germinating conidia-germinating EC and RSC, and secondary conidium on germ tube. b Means followed by same letter within the column are not significantly different (P . 0.05: Tukey’s studentized test). c Means separation for temperature within each substrate. NS, not significantly different (P . 0.05); * P , 0.05; ** P , 0.01; *** P , 0.001; H0 , coefficient, 0. d Means separation for time within each temperature and substrate. NS, not significantly different (P . 0.05); * P , 0.05; ** P , 0.01; *** P , 0.001; H0 , coefficient, 0.
role of topographical stimuli on the appressorial formation of M. anisopliae on Manduca sexta was reported by St. Leger et al. (1991). The leaf surface chemicals and pH on the tobacco leaf might also have made that surface similar to aphid cuticle. Observing the developmental morphology on coverslips treated with tobacco leaf surface extracts might provide further insight on this aspect. Decline in the virulence after successive subculturing in vitro has been reported in some species of entomopathogenic fungi including P. neoaphidis (Rockwood, 1950; Morrow et al., 1989; Hajek et al., 1990). Observance of only one penetration in our study might be the result of loss of virulence of P. neoaphidis due to in vitro subculturing. However, it is not known if this had any influence on its germination behavior. Influence of Temperature The time taken for P. neoaphidis to kill blue–green aphid, Acyrthosiphon kondoi, decreased with increase in temperature within the range of 8–20°C (Milner and Bourne, 1983). The temperature optima for in vitro
germination of primary and secondary conidia of P. neoaphidis is 18 to 23°C (Morgan et al., 1992, 1995). Since ellipsoid secondary conidia and ellipsoid primary conidia were grouped together, the proportion of primary conidia germinating and producing secondary conidia could not be determined in our study. However, 20°C seemed to be more favorable for infection than 13°C since appressorial formation was significantly higher at 20°C on aphid and leaf (Table 1). Correspondingly, round secondary conidia and germinating conidia, representing the successive stages of development, were less at 20 than at 13°C on these two surfaces, but the difference was not significant on aphid. Influence of Time Appressorial formation was similar at 12 and 24 h postinoculation on all surfaces at 20°C probably because this temperature was more favorable for infection (Table 1). At 13°C, P. neoaphidis took longer to produce appressoria as indicated by higher proportion of germinating conidia and lower proportion of appres-
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soria at 12 than at 24 h postinoculation. Some of the germinating conidia may have produced appressoria by 24 h postinoculation, resulting in lower proportions of germinating conidia and higher proportions of appressoria at that time. The proportion of round secondary conidia to ellipsoid conidia increased with increasing time at four temperatures between 10 and 30°C for P. neoaphidis cultured in vitro (Morgan et al., 1995). In this study, the proportion of round secondary conidia, among other structures found, was significantly higher with increase in time only at 20°C on coverslip and 13°C on leaf. The proportion of ellipsoid conidia significantly decreased at corresponding time and temperatures on those substrates. Since tobacco aphids, tobacco leaves, and inert substrates like soil are the most likely surfaces for conidia of P. neoaphidis to land on in a tobacco crop ecosystem, knowledge of pathogen development on these substrates could be helpful to understand disease dynamics. Further research on the influence of host proximity on conidial germination should be considered. ACKNOWLEDGMENTS We are grateful for the financial support provided by R. J. Reynolds Tobacco Company for conducting this study. We thank R. A. Humber, USDA-ARS, Ithaca, New York, for reviewing an earlier version of the manuscript.
REFERENCES Brobyn, P. J., and Wilding, N. 1977. Invasive and developmental processes of Entomophthora species infecting aphids. Trans. Br. Mycol. Soc. 69, 349–366. Brobyn, P. J., Wilding, N., and Clark, S. J. 1987. Laboratory observations on the effect of humidity on the persistence of infectivity of conidia of the aphid pathogen Erynia neoaphidis. Ann. Appl. Biol. 110, 579–584. Brown, G. C., Prochaska, G. L., Hildebrand, D. F., Nordin, G. L., and Jackson, D. M. 1995. Green leaf volatiles inhibit conidial germination of the entomopathogen Pandora neoaphidis (Entomophthorales: Entomophthoraceae). Environ. Entomol. 24, 1637–1643. Butt, T. M., Beckett, A., and Wilding, N. 1990. Histological study of the invasive and developmental processes of the aphid pathogen Erynia neoaphidis (Zygomycotina: Entomophthorales) in the pea aphid Acyrthosiphon pisum. Can. J. Bot. 68, 2153–2163. Carruthers, R. I., and Hural, K. 1990. Fungi as naturally occurring entomopathogens. In ‘‘New Direction in Biological Control: Alterna-
tives for Suppressing Agricultural Pests and Diseases’’ (P. Dunn and T. Baker, Eds.), pp. 115–138. A. R. Liss, New York. Fransen, J. J. 1995. Survival of spores of the entomopathogenic fungus Aschersonia aleyrodis (Deuteromycotina: Coelomycetes) on leaf surfaces. J. Invertebr. Pathol. 65, 73–75. Hajek, A. E., Humber, R. A., and Griggs, M. H. 1990. Decline in virulence of Entomophaga maimaiga (Zygomycetes: Entomophthorales) with repeated in vitro subculture. J. Invertebr. Pathol. 56, 91–97. Magalhaes, B. P., Humber, R. A., Shields, E. J., and Roberts, D. W. 1991. Effects of environment and nutrition on conidium germination and appressorium formation by Zoophthora radicans (Zygomycetes: Entomophthorales): A pathogen of the potato leafhopper (Homoptera: Cicadellidae). Environ. Entomol. 20, 1460–1468. Milner, R. J., and Bourne, J. 1983. Influence of temperature and duration of leaf wetness on infection of Acyrthosiphon kondoi with Erynia neoaphidis. Ann. Appl. Biol. 102, 19–27. Morgan, L. W., Boddy, L., and Wilding, N. 1992. The survival and germination of Erynia neoaphidis conidia. 25th Annual Meeting of Society for Invertebrate Pathology, August 16–21, Heidelberg, Germany. Morgan, L. W., Boddy, L., Clark, S. J., and Wilding, N. 1995. Influence of temperature on germination of primary and secondary conidia of Erynia neoaphidis (Zygomycetes: Entomophthorales). J. Invertebr. Pathol. 65, 132–138. Morrow, B. J., Boucias, D. G., and Heath, M. A. 1989. Loss of virulence in an isolate of an entomopathogenic fungus, Nomuraea rileyi, after serial in vitro passage. J. Econ. Entomol. 82, 404–407. Rockwood, L. P. 1950. Entomogenous fungi of the family Entomophthoraceae in the Pacific Northwest. J. Econ. Entomol. 43, 704–707. SAS Institute. 1994. ‘‘SAS User’s Guide: Statistics, Release 6.10.’’ SAS Institute, Cary, NC. St. Leger, R. J. 1993. Biology and mechanisms of insect-cuticle invasion by Deuteromycete fungal pathogens. In ‘‘Parasites and Pathogens of Insects’’ (N. E. Beckage, S. N. Thompson, and B. A. Federici, Eds.), pp. 211–229. Academic Press, New York. St. Leger, R. J., Butt, T. M., Goettel, M. S., Staples, R. C., and Roberts, D. W. 1989. Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13, 274–288. St. Leger, R. J., Goettel, M., Roberts, D. W., and Staples, R. C. 1991. Pre-penetration events during infection of host cuticle by Metarhizium anisopliae. J. Invertebr. Pathol. 57, 146–147. Wilding, N. 1971. Germination of conidia. Report of Rothamsted Experimental Station for 1970, Part 1, p. 206. Wilding, N., and Brady, B. L. 1984. Erynia neoaphidis. In ‘‘Commonwealth Mycological Institute, Descriptions of Pathogenic Fungi and Bacteria,’’ No. 815. The Cambrian News, UK. Yu, Z., Nordin, G. L., Brown, G. C., and Jackson, D. M. 1995. Studies on Pandora neoaphidis (Entomophthorales: Entomophthoraceae) infectious to the red morph of tobacco aphid (Homoptera: Aphididae). Environ. Entomol. 24, 962–966.
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Influence of Substrate Type and Temperature on the Developmental Morphology of Pandora neoaphidis (Zygomycetes: Entomophthorales), a Pathogen of the Tobacco Aphid (Homoptera: Aphididae) Surendra K. Dara1 and Paul J. Semtner Southern Piedmont Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University, Route 3 Box 60, Blackstone, Virginia 23824 Received November 4, 1996; accepted February 5, 1998
Developmental morphology of Pandora neoaphidis was observed on the surfaces of the tobacco aphid, Myzus nicotianae, tobacco leaves (Nicotiana tabacum), and glass coverslips at 13 and 20°C for 12 and 24 h postinoculation. Pandora neoaphidis responded similarly on the two living substrates, but differed on the inert coverslips. The proportions of ellipsoid conidia (primary and secondary) were similar on all substrates. Higher proportions of appressoria and lower proportions of round secondary conidia and germinating conidia occurred on the aphids and leaves than on the coverslips. Appressoria predominated over round secondary conidia and germinating conidia on the living substrates at 20°C, but the opposite was seen at 13°C. The proportions of ellipsoid conidia were similar at both temperatures. On coverslips, the proportions of appressoria and round secondary conidia were similar at both temperatures. However, the proportions of germinating and ellipsoid conidia were higher at 13 and 20°C, respectively. r 1998 Academic Press Key Words: Pandora neoaphidis; Myzus nicotianae; substrate; temperature; germination; tobacco leaf.
The fate of conidia after they land on the host surface determines the success of invasion by the pathogen. Brobyn and Wilding (1977) and Butt et al. (1990) studied the invasive and developmental processes of P. neoaphidis on Acyrthosiphon pisum. The primary conidium of P. neoaphidis germinates about 4 h postinoculation and forms a germ tube, an infective appressorium, or a secondary conidium (Brobyn and Wilding, 1977; Butt et al., 1990). Differential responses have been demonstrated for P. neoaphidis in conidial germination on coverslips and leaves of field beans (Brobyn et al., 1987). Fransen (1995) demonstrated the response of Aschersonia aleyrodis, another entomopathogen, on cucumber leaves and the cuticle of Trialeurodes vaporariorum (Fransen, 1995). Information on the response of P. neoaphidis to the type of substrate will provide a greater understanding of the fate of inoculum on different surfaces in the environment. This research examined the developmental morphology of conidia of P. neoaphidis inoculated on three surfaces and incubated at two temperatures. MATERIALS AND METHODS
INTRODUCTION
Rearing Aphids
The host range of Pandora neoaphidis includes several species of aphids and a few species of hemipterans (Wilding and Brady, 1984). Its germination, type of conidia, growth in vitro, and production of conidia in vivo are influenced by temperature (Morgan et al., 1995; Yu et al., 1995). Optimal temperatures for conidial germination of P. neoaphidis are between 18 and 23°C (Morgan et al., 1992, 1995), while maximum conidial production occurs between 10 and 25°C (Yu et al., 1995). 1Current address: International Institute of Tropical Agriculture, 08 BP 0932 Tri Postal, Cotonou, Republic of Benin, West Africa. E-mail: [email protected].
0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
Tobacco aphids, Myzus nicotianae, were collected from field tobacco, Nicotiana tabacum, and reared on tobacco plants growing in sterile potting medium in a growth chamber at 22°C and 16L:8D photophase. Newly born aphids were collected from these plants with a camel’s hair brush and transferred to excised leaves from tobacco grown in the greenhouse. The leaves were rinsed with deionized water and air dryed. These leaves were placed in Styrofoam cups (710 ml) and their petioles were inserted in 1% water agar (Fisher Scientific, Springfield, NJ) in the bottom of the cups. Lids were placed on the cups to keep the aphids inside. Aphids were transferred to new leaves after 5 or 6 days. When they were 9 days old, aphids were again trans-
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ferred to new leaves in new cups and allowed to deposit nymphs for 6–8 h. Viviparae were then removed using a moist camel’s hair brush and neonate nymphs were retained in the cups. Nymphs from these cultures were used in the experiment when they were 5 days old. Culturing P. neoaphidis To culture P. neoaphidis, infected tobacco aphids were collected from the field, surface sterilized for 30 to 60 s each in a 5–10% bleach (sodium hypoclorite 5.25%) and a 0.01% gentamicin solution, and transferred onto modified SEMA medium (G. L. Nordin personal communication; Yu et al., 1995). Ingredients of SEMA included 74.6% fresh yolk from unwashed eggs, 14.4% vitamin D milk, 11% Sabouraud maltose agar (Difco Laboratories, Detroit, MI), 0.1% gentamicin (Sigma Chemical Co., St. Louis, MO), and 0.05% penicillin (Agri Laboratories Ltd., St. Joseph, MO). These cultures were incubated at 20°C and 14L:10D photophase. The pathogen used for the experiment had been subcultured four times in vitro after it was isolated from the aphid host. Two- to 3-week-old cultures from a common source were used for the experiment. Sporulating cultures were selected for the experiment based on the conidial deposition on the lids of Petri plates. Inoculating Aphids, Tobacco Leaves, and Coverslips On the day of the experiment, 25 aphids, 5 days old, were placed on a separate tobacco leaf disc (about 55 mm dia) in each of four small Petri plates (60 3 15 mm). Before the discs were cut, tobacco leaves from greenhouse plants were rinsed with deionized water. A filter paper (Whatman No. 1, 55-mm circle) was placed on the bottom and a similar one was attached to the lid of each Petri plate with nontoxic Permanent Glue Stic (Avery Dennison, Framingham, MA). These filter papers were moistened to maintain the high humidity ($98%) required for conidial germination (Wilding, 1971; Carruthers and Hural, 1990; Yu et al., 1995). In each Petri plate, five small pieces of coverslip (about 40 mm2 ) were placed on the leaf disc, one in the center and one in each of four outer quadrants. Large Petri plates (100 3 15 mm) containing fungal cultures were inverted over the small plates to expose aphids, leaf discs, and coverslips to conidial showers from the cultures. The plates were then covered with moist paper towels, placed in a sealed plastic bag, and inoculated in the dark at 20°C. Relative humidity inside the plastic bags was measured with a hygrometer (127 mm diam by 32 mm) (Abbeon Cal. Inc., Santa Barbara, CA). Moist paper towels helped to maintain the relative humidity at $98% in the plastic bags. Aphids were exposed to conidial showers for 4 h. During inoculation, culture plates were rotated 90° every 60 min to promote uniform conidial showers over
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the exposed area. After inoculation, cultures were removed and lids were placed on the small plates. Two plates each were incubated in the dark at 13 and 20°C. At 12 and 24 h postinoculation, one plate each was taken from the 13 and 20°C chambers, respectively. The aphids from each Petri plate were divided into five groups of five aphids. Five small pieces (about 40 mm2 ) were cut from the tobacco leaf disc in each Petri plate to represent five samples of each treatment. Thus each temperature and time treatment (Petri plate) contained five groups of aphids and five pieces each of tobacco leaf and coverslip. A group of five aphids, a piece of tobacco leaf, and a piece of coverslip represented a replication from a section of a leaf disk. SEM The surfaces of aphids, pieces of tobacco leaf, and coverslips were critical point dried and sputter-coated with gold–palladium (10 nm) for scanning electron microscopy (SEM). The conidial germination was observed under SEM (Philips 505) on the surfaces of tobacco aphid, tobacco leaf, and coverslip. Observations were made on the entire dorsal region of each tobacco aphid (ca. 1 to 2 mm2 ) and at five random locations (area of each location about 0.2 mm2 ) on each piece of tobacco leaf and coverslip. Statistical Analysis Analysis of variance (GLM procedure, SAS Institute, 1994) was used to analyze all data. Significantly different means for substrate were separated by Tukey’s studentized range test (SAS Institute, 1994). Surface effects were analyzed separately within each temperature 3 time treatment. In addition, comparisons were made for treatments over time and over temperature. The proportions of different categories of structures found on three substrates were calculated. Data were arcsine-transformed to stabilize variances. RESULTS
Conidial germination as indicated by germ tubes, appressoria, or secondary conidia occurred on all substrates. Structures found on the three substrates were placed in the following categories: nongerminated ellipsoid conidium (Fig. 1), germinated conidium with collapsed spore and intact germ tube (Fig. 2), conidium that produced an appressorium (Fig. 3), conidium with penetration peg (Fig. 4), round secondary conidium (Fig. 5), secondary conidium developing on a germ tube (Fig. 6), ellipsoid conidium germinating by means of germ tube or initiation of successive (secondary or tertiary) conidium or both (Fig. 7), and round secondary conidium germinating by means of germ tube (Fig. 8). Primary conidia and secondary conidia that resembled
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primary conidia were considered as ellipsoid conidia since the two types of conidia were often difficult to separate. Germination of conidia was influenced by substrate, temperature, and time (Table 1). The relative proportion of different structures formed during germination varied on the three substrates and at the two temperatures when observed at two time intervals (P , 0.05).
interaction: df 2, F 5 6.73, P 5 0.0031). Time had no effect at either temperature on the proportion of appressoria on aphids and at 20°C on coverslips and leaves. However, at 13°C significantly higher (P 5 0.01) proportions of appressoria were found at 24 h than at 12 h postinoculation on coverslips and leaves (temperature 3 time interaction: df1, F 5 6.35, P 5 0.0160). Ellipsoid Conidium
Germ Tube The proportions of germ tubes observed on the three substrates were not significantly different (results not shown). Temperature did not influence germ tubes on aphids and coverslips, but significantly higher (P 5 0.0001) proportions of germ tubes were found on leaves at 20 than at 13°C. Time did not influence the formation of germ tubes on aphids, but significantly higher (P , 0.05) proportions of germ tubes were seen at 24 h than at 12 h postinoculation for both temperatures on leaves and coverslips.
The overall proportion of nongerminated ellipsoid conidia was similar on all substrates (Table 1). However, the proportion was significantly higher on coverslips (P 5 0.0002) at 20 than at 13°C. Time had no influence on ellipsoid conidia at both temperatures on aphids, at 13°C on coverslips, and at 20°C on leaves. The proportion of ellipsoid conidia significantly decreased (P 5 0.01) 24 h postinoculation at 20°C on coverslips and at 13°C on leaves (surface 3 temperature 3 time interaction: df 2, F 5 4.34, P 5 0.0199). Round Secondary Conidium
Penetration Peg Penetration was seen at 20°C on the abdomen of an aphid within 12 h postinoculation. Since there was only one penetration peg in the entire study, it was excluded from the analysis. Germinating Round Secondary Conidium Germinating round secondary conidia were found only 24 h postinoculation at both temperatures on coverslips and at 20°C on leaves. They were not seen on aphids. Secondary Conidium on Germ Tube Germ tubes bearing secondary conidia were not seen on aphids. They occurred on coverslips at both temperatures and on leaves at 20°C. However, germ tubes were not observed within 12 h postinoculation on coverslips at 13°C and on leaves at 20°C. Since no sign of successive development was seen in germ tubes with collapsed conidia, the remaining structures were regrouped into four categories viz., appressoria, ellipsoid conidia, round secondary conidia, and germinating conidia (including germinating ellipsoid and round secondary conidia and conidia on germ tubes) and compared (Table 1). Appressorium Pandora neoaphidis produced similar proportions of appressoria on aphids and leaves, but proportions were significantly lower (P 5 0.0001) on the inert coverslips (Table 1). Formation of appressoria was significantly higher (P , 0.05) at 20 than at 13°C on aphids and leaves, but not on coverslips (surface 3 temperature
The majority of round secondary conidia on all substrates were still attached to the collapsed primary conidia where they were produced. The production of round secondary conidia was significantly higher (P 5 0.002) on coverslips than on aphids or leaves (Table 1). Proportions of round secondary conidia were influenced by temperature on aphids and leaves, but not on coverslips. On leaves and aphids, significantly lower (P 5 0.0001) proportions of round secondary conidia were seen at 20 than at 13°C (surface 3 temperature interaction: df 2, F 5 6.64, P 5 0.0033). A significantly higher proportion of round secondary conidia was seen at 24 h than at 12 h postinoculation at 20°C on coverslips, and at 13°C on leaves (surface temperature 3 time interaction: df 2, F 5 6.6, P 5 0.0034). Germinating Conidium Significantly higher proportions of germinating ellipsoid conidia were seen on coverslips than on aphids and leaves, and at 13 than at 20°C on all substrates (surface 3 temperature interaction: df 2, F 5 3.5, P 5 0.04). The proportion of germinating ellipsoid conidia was higher at 12 h than at 24 h postinoculation on all substrates at 13°C; however, the differences were not significant on aphids (temperature 3 time interaction: df1, F 5 20.68, P 5 0.0001). Similar proportions of germinating ellipsoid conidia were seen on all substrates at both time intervals at 20°C. DISCUSSION
Stimuli from the substrate induce formation of appressoria in entomopathogenic fungi (St. Leger, 1993). Our observations indicate that cues from the living
SUBSTRATE AND TEMPERATURE EFFECTS ON Pandora neoaphidis
FIGS. 1–4.
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Scanning electron micrographs.
substrates are important in the formation of appressoria in P. neoaphidis. The proportions of appressoria were significantly higher on the living substrates than on the inert coverslips. The production of appressoria in P. neoaphidis precedes penetration of the host cuticle and is in response to the recognition that the substrate is suitable for infection (Butt et al., 1990). Other entomopathogenic fungi form appressoria in response to nutritional and physical stimuli. Various levels of nitrogenous nutrients, glucose, and hard, hydrophobic surfaces induce in vitro formation of appressoria in Metarhizium anisopliae (St. Leger et al., 1989; St. Leger, 1993). Appressorial formation by Zoophthora radicans is severely depressed in vitro in the absence of nitrogen and carbon sources (Magalhaes et al., 1991). When conditions are not favorable for infection, the fungus continues to produce successive stages of conidia, until the nutrients are depleted. In our study,
P. neoaphidis expressed this behavior by producing significantly higher proportions of round secondary conidia and germinating conidia and less of appressoria on the inert coverslips. In contrast, the pathogen produced more infective appressoria and less successive stages of development on aphid and leaf surfaces, while the proportion of ellipsoid conidia remained constant on all substrates. Apparently P. neoaphidis did not receive the signal to produce infective structures on coverslips and continued to germinate by other means. Brobyn et al. (1987) reported that conidia of P. neoaphidis rapidly lost their infectivity at 100% relative humidity on coverslips due to exhaustion of energy reserves from production of successive generations of secondary conidia in a short period. The conidia of A. aleyrodis, another entomopathogenic fungus, had lower germination on the surface of cucumber leaf than on the cuticle of fourth-instar
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FIG. 1. Ellipsoid primary or secondary conidia on aphid cuticle (bar 5 10 µm). FIG. 2. Germinated conidium with collapsed spore (arrowhead) and intact germ tube (arrow) on coverslip (bar 5 100 µm). FIG. 3. Conidium (arrowhead) with globose appressorium (arrow) on aphid cuticle (bar 5 10 µm). FIG. 4. Conidium (arrowhead) with penetration peg (arrow) on the abdomen of aphid (bar 5 10 µm). FIGS. 5–8. Scanning electron micrographs. FIG. 5. Round secondary conidium (arrow) singly and on collapsed primary conidium (arrowhead) on coverslip (bar 5 10 µm). FIG. 6. Secondary conidium (arrow) developing on a germ tube (arrowhead) on coverslip (bar 5 10 µm). FIG. 7. Germinating ellipsoid conidia. (A) Ellipsoid conidia (arrowhead) with different stages of development of secondary/tertiary conidia (arrow) (bar 5 10 µm), (B) ellipsoid conidium with germ tube (bar 5 10 µm), (C) ellipsoid conidium with initiation of secondary/ tertiary conidium and germ tube (bar 5 10 µm) on aphid cuticle. FIG. 8. Germinating round secondary conidium, on coverslip (bar 5 100 µm). Round secondary conidium (arrowhead) with a germ tube (arrow) on a collapsed primary conidium.
larvae of greenhouse whitefly, T. vaporariorum (Fransen, 1995). This reduced germination was attributed to lack of stimulation or presence of inhibitory factors on cucumber leaves. Germination of P. neoaphidis conidia was slower on leaves of field beans (Vicia faba) than on coverslips at 70–77% relative humidity (Brobyn et al., 1987). Brown et al. (1995) reported from in vitro experiments that conidial germination of P. neoaphidis was inhibited by volatiles from tobacco leaves, especially if the tobacco leaf has supported a previous infestation of tobacco aphids. They also re-
ported that aphid infection rates were not affected by green leaf volatiles. These results led them to hypothesize that there is little inhibition when the conidia are in direct contact with the aphid cuticle. In our study, conidial germination was similar on aphid cuticle and tobacco leaf. Unknown factors may have stimulated appressorial formation or simulated aphid cuticle on tobacco leaf surface. Similarities between the convoluted topography of aphid cuticle and tobacco leaf surface may have caused the similar responses of P. neoaphidis on those two surfaces. The
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TABLE 1 Relative Proportion of Appressoria, Ellipsoid Conidia, Round Secondary Conidia, and Germinating Conidia Mean 6 SE Effect
Temp. (°C)
Time
APP a
EC
RSC
GC
Substrate b Aphid Coverslip Leaf Temperature c Aphid Aphid Coverslip Coverslip Leaf Leaf Time d Aphid Aphid Aphid Aphid Coverslip Coverslip Coverslip Coverslip Leaf Leaf Leaf Leaf
13 20 13 20 13 20 13 13 20 20 13 13 20 20 13 13 20 20
12 24 12 24 12 24 12 24 12 24 12 24
22.6 6 4.3a 4.6 6 1.4b 22.0 6 3.8a
54.3 6 3.9a 50.2 6 3.4a 56.6 6 3.0a
9.9 6 1.9b 16.4 6 2.2a 8.7 6 2.3b
13.3 6 3.5b 28.9 6 2.7a 12.8 6 2.1b
14.0 6 4.1** 35.5 6 5.7 4.5 6 1.7NS 4.7 6 2.3 11.5 6 3.9** 32.5 6 4.5
50.9 6 5.1NS 59.3 6 5.9 40.8 6 2.0*** 59.6 6 5.1 53.1 6 4.0NS 60.1 6 4.4
13.2 6 2.3** 5.0 6 1.7 16.5 6 2.6NS 16.2 6 3.7 16.7 6 2.8*** 0.7 6 0.2
22.0 6 3.4*** 0.2 6 0.2 38.3 6 2.2*** 19.5 6 2.5 18.8 6 3.0*** 6.8 6 1.1
7.2 6 2.5NS 22.4 6 6.8 37.1 6 11.8NS 33.9 6 4.5 0.2 6 0.1* 8.7 6 1.8 0.9 6 0.5NS 8.6 6 3.9 0.9 6 0.2** 22.1 6 3.4 33.7 6 8.4NS 31.3 6 4.5
50.7 6 2.5NS 51.1 6 12.0 61.2 6 12.5NS 57.5 6 3.8 39.5 6 1.8NS 42.0 6 3.6 72.9 6 3.9** 46.3 6 3.6 61.8 6 5.0* 44.4 6 3.4 60.7 6 7.6NS 59.4 6 5.5
13.6 6 3.7NS 12.6 6 3.1 1.3 6 0.7NS 8.6 6 0.9 17.8 6 1.6NS 15.2 6 5.2 7.4 6 2.1** 25.0 6 4.4 11.1 6 2.4* 22.3 6 3.7 0.4 6 0.2NS 0.9 6 0.4
28.5 6 1.6NS 13.9 6 4.9 0.4 6 0.4NS 0.0 6 0.0 42.4 6 2.4NS 34.1 6 2.6 18.8 6 3.5NS 20.2 6 3.8 26.3 6 2.9* 11.2 6 2.2 5.2 6 1.2NS 8.3 6 1.7
a APP, appressorium 1 penetration; EC, ellipsoid primary or secondary conidium; RSC, round secondary conidium; GC, germinating conidia-germinating EC and RSC, and secondary conidium on germ tube. b Means followed by same letter within the column are not significantly different (P . 0.05: Tukey’s studentized test). c Means separation for temperature within each substrate. NS, not significantly different (P . 0.05); * P , 0.05; ** P , 0.01; *** P , 0.001; H0 , coefficient, 0. d Means separation for time within each temperature and substrate. NS, not significantly different (P . 0.05); * P , 0.05; ** P , 0.01; *** P , 0.001; H0 , coefficient, 0.
role of topographical stimuli on the appressorial formation of M. anisopliae on Manduca sexta was reported by St. Leger et al. (1991). The leaf surface chemicals and pH on the tobacco leaf might also have made that surface similar to aphid cuticle. Observing the developmental morphology on coverslips treated with tobacco leaf surface extracts might provide further insight on this aspect. Decline in the virulence after successive subculturing in vitro has been reported in some species of entomopathogenic fungi including P. neoaphidis (Rockwood, 1950; Morrow et al., 1989; Hajek et al., 1990). Observance of only one penetration in our study might be the result of loss of virulence of P. neoaphidis due to in vitro subculturing. However, it is not known if this had any influence on its germination behavior. Influence of Temperature The time taken for P. neoaphidis to kill blue–green aphid, Acyrthosiphon kondoi, decreased with increase in temperature within the range of 8–20°C (Milner and Bourne, 1983). The temperature optima for in vitro
germination of primary and secondary conidia of P. neoaphidis is 18 to 23°C (Morgan et al., 1992, 1995). Since ellipsoid secondary conidia and ellipsoid primary conidia were grouped together, the proportion of primary conidia germinating and producing secondary conidia could not be determined in our study. However, 20°C seemed to be more favorable for infection than 13°C since appressorial formation was significantly higher at 20°C on aphid and leaf (Table 1). Correspondingly, round secondary conidia and germinating conidia, representing the successive stages of development, were less at 20 than at 13°C on these two surfaces, but the difference was not significant on aphid. Influence of Time Appressorial formation was similar at 12 and 24 h postinoculation on all surfaces at 20°C probably because this temperature was more favorable for infection (Table 1). At 13°C, P. neoaphidis took longer to produce appressoria as indicated by higher proportion of germinating conidia and lower proportion of appres-
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soria at 12 than at 24 h postinoculation. Some of the germinating conidia may have produced appressoria by 24 h postinoculation, resulting in lower proportions of germinating conidia and higher proportions of appressoria at that time. The proportion of round secondary conidia to ellipsoid conidia increased with increasing time at four temperatures between 10 and 30°C for P. neoaphidis cultured in vitro (Morgan et al., 1995). In this study, the proportion of round secondary conidia, among other structures found, was significantly higher with increase in time only at 20°C on coverslip and 13°C on leaf. The proportion of ellipsoid conidia significantly decreased at corresponding time and temperatures on those substrates. Since tobacco aphids, tobacco leaves, and inert substrates like soil are the most likely surfaces for conidia of P. neoaphidis to land on in a tobacco crop ecosystem, knowledge of pathogen development on these substrates could be helpful to understand disease dynamics. Further research on the influence of host proximity on conidial germination should be considered. ACKNOWLEDGMENTS We are grateful for the financial support provided by R. J. Reynolds Tobacco Company for conducting this study. We thank R. A. Humber, USDA-ARS, Ithaca, New York, for reviewing an earlier version of the manuscript.
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